Generation of modified polymerases for improved accuracy in single molecule sequencing

[0001] This application is a non-provisional utility patent application claiming priority to and benefit of provisional patent application U.S. Ser. No. 61/278,041, filed Sep. 30, 2009, entitled “GENERATION OF MODIFIED POLYMERASES FOR IMPROVED ACCURACY IN SINGLE MOLECULE SEQUENCING” by Sonya Clark et al., and is a continuation-in-part of U.S. Ser. No. 12/384,112, filed Mar. 30, 2009, entitled “GENERATION OF MODIFIED POLYMERASES FOR IMPROVED ACCURACY IN SINGLE MOLECULE SEQUENCING” by Sonya Clark et al., which claims priority to and benefit of the following prior provisional patent applications: U.S. Ser. No. 61/072,645, filed Mar. 31, 2008, entitled “GENERATION OF POLYMERASES WITH IMPROVED CLOSED COMPLEX STABILITY AND DECREASED BRANCHING RATE” by Sonya Clark et al., and U.S. Ser. No. 61/094,843, filed Sep. 5, 2008, entitled “ENGINEERING POLYMERASES FOR MODIFIED INCORPORATION PROPERTIES” by Pranav Patel et al. Each of these applications that was cited in this application at the time of its filing is incorporated herein by reference in its entirety for all purposes.

[0002] The invention relates to modified DNA polymerases for single molecule sequencing. The polymerases include modified recombinant polymerases that display a reduction in the formation of branching fraction during single molecule sequencing for various nucleotide analogs, modified polymerases that display increased stability of closed polymerase-DNA complexes and enhanced polymerase processivity, and modified polymerases that exhibit one or more slow steps in their catalytic cycle. The invention also relates to methods for determining the sequence of nucleic acid molecules using such polymerases.

[0003] DNA polymerases replicate the genomes of living organisms. In addition to this central role in biology, DNA polymerases are also ubiquitous tools of biotechnology. They are widely used, e.g., for reverse transcription, amplification, labeling, and sequencing, all central technologies for a variety of applications such as nucleic acid sequencing, nucleic acid amplification, cloning, protein engineering, diagnostics, molecular medicine, and many other technologies.

[0004] Because of the significance of DNA polymerases, they have been extensively studied. This study has focused, e.g., on phylogenetic relationships among polymerases, structure of polymerases, structure-function features of polymerases, and the role of polymerases in DNA replication and other basic biology, as well as ways of using DNA polymerases in biotechnology. For a review of polymerases, see, e.g., Hiibscher et al. (2002) “Eukaryotic DNA Polymerases” Annual Review of Biochemistry Vol. 71: 133-163, Alba (2001) “Protein Family Review: Replicative DNA Polymerases” Genome Biology 2(1): reviews 3002.1-3002.4, Steitz (1999) “DNA polymerases: structural diversity and common mechanisms” J Biol Chem 274:17395-17398, and Burgers et al. (2001) “Eukaryotic DNA polymerases: proposal for a revised nomenclature” J Biol Chem. 276(47): 43487-90. Crystal structures have been solved for many polymerases, which often share a similar architecture. The basic mechanisms of action for many polymerases have been determined.

[0005] A fundamental application of DNA technology involves various labeling strategies for labeling a DNA that is produced by a DNA polymerase. This is useful in DNA sequencing, microarray technology, SNP detection, cloning, PCR analysis, and many other applications. Labeling is often performed in various post-synthesis hybridization or chemical labeling schemes, but DNA polymerases have also been used to directly incorporate various labeled nucleotides in a variety of applications, e.g., via nick translation, reverse transcription, random priming, amplification, the polymerase chain reaction, etc. See, e.g., Giller et al. (2003) “Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group-labeled 2′-deoxyribonucleoside-5′-triphosphates” Nucleic Acids Res. 31(10): 2630-2635, Augustin et al. (2001) “Progress towards single-molecule sequencing: enzymatic synthesis of nucleotide-specifically labeled DNA” J. Biotechnol. 86:289-301, Tonon et al. (2000) “Spectral karyotyping combined with locus-specific FISH simultaneously defines genes and chromosomes involved in chromosomal translocations” Genes Chromosom. Cancer 27:418-423, Zhu and Waggoner (1997) “Molecular mechanism controlling the incorporation of fluorescent nucleotides into DNA by PCR.” Cytometry, 28:206-211, Yu et al. (1994) “Cyanine dye dUTP analogs for enzymatic labeling of DNA probes” Nucleic Acids Res., 22:3226-3232, Zhu et al. (1994) “Directly labeled DNA probes using fluorescent nucleotides with different length linkers” Nucleic Acids Res. 22:3418-3422, and Reid et al. (1992) “Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy” Proc. Natl. Acad. Sci. USA, 89:1388-1392.

[0006] DNA polymerase mutants have been identified that have a variety of useful properties, including altered nucleotide analog incorporation abilities relative to wild-type counterpart enzymes. For example, Vent^A488L DNA polymerase can incorporate certain non-standard nucleotides with a higher efficiency than native Vent DNA polymerase. See Gardner et al. (2004) “Comparative Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase” J. Biol. Chem., 279(12), 11834-11842 and Gardner and Jack “Determinants of nucleotide sugar recognition in an archaeon DNA polymerase” Nucleic Acids Research, 27(12) 2545-2553. The altered residue in this mutant, A488, is predicted to be facing away from the nucleotide binding site of the enzyme. The pattern of relaxed specificity at this position roughly correlates with the size of the substituted amino acid side chain and affects incorporation by the enzyme of a variety of modified nucleotide sugars.

[0007] Additional modified polymerases, e.g., modified polymerases that display improved properties useful for single molecule sequencing (SMS) and other polymerase applications (e.g., DNA amplification, sequencing, labeling, detection, cloning, etc.), are desirable. The present invention provides new DNA polymerases with improved kinetic properties including reduced branching fraction formation, increased stability of closed polymerase-DNA complexes and increased processivity, and reduced rates for one or more steps in the catalytic cycle. Also included are methods of making such polymerases, methods of using such polymerases, and many other features that will become apparent upon a complete review of the following.

[0008] Modified DNA polymerases can find use in such applications as, e.g., single-molecule sequencing (SMS), genotyping analyses such as SNP genotyping using single-base extension methods, and real-time monitoring of amplification, e.g., RT-PCR. The invention provides compositions comprising recombinant polymerases that comprise mutations which confer properties which can be particularly desirable for these applications. These improved properties can, e.g., facilitate readout accuracy and improve polymerase processivity and other kinetic parameters. Also provided by the invention are methods of generating such modified polymerases and methods in which such polymerases can be used to, e.g., sequence a DNA template and/or make a DNA.

[0009] Mutations are typically described herein with reference to an amino acid and corresponding position relative to a wild-type Φ29 DNA polymerase (SEQ ID NO:1). For example, it will be understood that, e.g., an L253 mutation indicates an amino acid substitution or deletion of the leucine residue at position 253 relative to a wild-type Φ29 DNA polymerase. For example, an L253 mutation can be a substitution of alanine for leucine at position 253, designated L253A. This nomenclature applies to each mutation or combinations described herein. As an additional example, a P477 mutation refers to an amino acid substitution or deletion of the proline residue at position 477 relative to a wild-type Φ29 DNA polymerase, while a P477Q mutation refers to a substitution of glutamine for proline at position 477. A mutation at a particular residue/position can also refer to an insertion of one or more amino acids immediately following that residue/position in the protein, as described further hereinbelow.

[0010] In one aspect, the invention provides compositions that include a recombinant DNA polymerase homologous to a Φ29 DNA polymerase. The recombinant DNA polymerase can include a mutation or combination of mutations relative to a wild-type Φ29 DNA polymerase (SEQ ID NO:1) selected from: an L253 mutation, where the polymerase further comprises a mutation at one or more of T368, E375, A484, or K512; an E375 and K512 mutation, where the polymerase further comprises a mutation at one or more of L253, T368 or A484; an 193 mutation; an S215 mutation; an E420 mutation; a P477 mutation; a D66R mutation; a K135R mutation; a K138R mutation; an L253T mutation; a Y369G mutation; a Y369L mutation; an L384M mutation; a K422A mutation; an I504R mutation; an E508K mutation; an E508R mutation; a D510K mutation; at least one mutation or combination of mutations selected from those listed in Tables 6, 9 and 10; and a T368 mutation, where the polymerase further comprises a mutation at one or more of E375 or K512. The polymerase can optionally include a mutation that inhibits exonuclease activity of the polymerase, e.g., an N62 mutation, a D12 mutation, a D66 mutation, a T15 mutation, etc.

[0011] When the polymerase includes an L253 mutation and a mutation at one or more of T368, E375, A484 or K512, the polymerase optionally includes E375Y and K512Y mutations.

[0012] Polymerases of the invention can include a set of mutations selected from: (N62D, L253A, E375Y, E420M, A484E, and K512Y); (N62D, L253A, E375Y, K422A, A484E, and K512Y); (N62D, L253A, E375Y, A484E, E508K, and K512Y); (N62D, S215D, L253A, E375Y, A484E, and K512Y); (N62D, L253T, E375Y, A484E, and K512Y); (N62D, L253A, Y369H, E375Y, A484E, and K512Y); (N62D, L253A, Y369G, E375Y, A484E, and K512Y); (N62D, L253A, Y369L, E375Y, A484E, and K512Y); (N62D, L253A, E375F, A484E, and K512Y); (D66R, L253A, E375Y, A484E, and K512Y); (N62D, L253A, E375Y, A484E, I504R, and K512Y); (N62D, L253A, E375Y, A484E, D510K, and K512Y); (L253A, E375Y, A484E, and K512Y); (L253A, E375Y, A484E, and K512Y); (L253A, E375Y, A484E, E508R, and K512Y); (N62D, E375Y, and K512Y); (N62D, T368F, E375Y, and K512Y); (N62D, T368F, E375Y, and K512Y); (T368F, E375Y, and K512Y); (N62D, L253A, E375Y, A484E, and K512Y); (N62D, L253A, E375Y, and K512Y); (N62D, T368F, E375Y, A484E, and K512Y.co); (N62D, T368F, E375Y, A484E, and K512Y); (N62D, L253A, E375Y, A484E, and K512Y.co); (N62D, L253A, E375Y, A484E, and K512Y.co); (N62D, H149M, T368F, E375Y, D510M, K512Y, and D523M.co); (N62H, E375Y, A484E, E508R, and K512Y.co); (D12R, N62H, T368F, E375Y, A484E, and K512Y.co); (D12R, T368F, E375Y, A484E, E508R, 511.1K, 511.2S, 512.1G, and 512.2S.co); (D12R, T368F, E375Y, 1378W, A484E, E508R, 511.1K, 511.2S, 512.1G, and 512.2S.co); (Y148A, E375Y, A484E, and K512Y.co); (N62D, A190E, E375Y, K422A, A484E, E508R, and K512Y.co); (N62D, 193Y, T368F, T372Y, E375Y, 1378W, K478Y, A484E, E508R, 511.1K, 511.2S, K512Y, 512.1G, and 512.2S.co); (N62D, T368F, E375Y, P477Q, A484E, and K512Y); (N62D, T368F, E375Y, L384M, A484E, and K512Y.co); (T368F, E375Y, P477E, and K512Y); (A176V, T368F, E375Y, and K512Y); (T368F, E375Y, K422R, and K512Y); (N62D, E375Y, P477Q, A484E, and K512Y.co); (193F, 368F, E375Y, A484E, and K512Y); and (Y148A, E375Y, A484E, and K512Y.co).

[0013] Optionally, polymerases of the invention that include a particular set of mutations can further include one or more exogenous or heterologous features at the N-terminal and/or C-terminal region of the polymerase. For example, a polymerase of the invention can include the following exogenous or heterologous features and sets of mutations selected from: (N-terminal biotinylation tag, N-terminal His10 tag, N62D, L253A, E375Y, E420M, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, L253A, E375Y, K422A, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, L253A, E375Y, A484E, E508K, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, S215D, L253A, E375Y, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, L253T, E375Y, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, L253A, Y369H, E375Y, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, L253A, Y369G, E375Y, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, L253A, Y369L, E375Y, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, L253A, E375F, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, D66R, L253A, E375Y, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, L253A, E375Y, A484E, I504R, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, L253A, E375Y, A484E, D510K, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, C-terminal His10 tag, L253A, E375Y, A484E, and K512Y); (N-terminal biotinylation tag V7, N-terminal His10 tag, C-terminal His10 tag, L253A, E375Y, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, C-terminal His10 tag, L253A, E375Y, A484E, E508R, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N-terminal Xa tag, N62D, E375Y, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N-terminal Xa tag, N62D, T368F, E375Y, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N-terminal Xa tag, N62D, T368F, E375Y, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N-terminal Xa tag, T368F, E375Y, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, L253A, E375Y, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, L253A, E375Y, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, C-terminal His10 tag, N62D, T368F, E375Y, A484E, and K512Y.co); (N-terminal biotinylation tag, N-terminal His10 tag, N-terminal Xa tag, N62D, T368F, E375Y, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, C-terminal His10 tag, N62D, L253A, E375Y, A484E, and K512Y.co); (N-terminal biotinylation tag, N-terminal His10 tag, C-terminal 1942link, C-terminal Ala10 tag, N62D, L253A, E375Y, A484E, and K512Y.co); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, H149M, T368F, E375Y, D510M, K512Y, and D523M.co); (N-terminal biotinylation tag, N-terminal His10 tag, C-terminal His10 tag, N62H, E375Y, A484E, E508R, and K512Y.co); (N-terminal biotinylation tag, N-terminal His10 tag, C-terminal His-10 tag, D12R, N62H, T368F, E375Y, A484E, and K512Y.co); (N-terminal biotinylation tag, N-terminal His10 tag, C-terminal His10 tag, D12R, T368F, E375Y, A484E, E508R, 511.1K, 511.2S, 512.1G, and 512.2S.co); (N-terminal biotinylation tag, N-terminal His10 tag, C-terminal His10 tag, D12R, T368F, E375Y, 1378W, A484E, E508R, 511.1K, 511.2S, 512.1G, and 512.2S.co); (N-terminal biotinylation tag, N-terminal His10 tag, Y148A, E375Y, A484E, and K512Y.co); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, A190E, E375Y, K422A, A484E, E508R, and K512Y.co); (N-terminal biotinylation tag, N-terminal His10 tag, C-terminal His10 tag, N62D, 193Y, T368F, T372Y, E375Y, 1378W, K478Y, A484E, E508R, 511.1K, 511.2S, K512Y, 512.1G, and 512.2S.co); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, T368F, E375Y, P477Q, A484E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, T368F, E375Y, L384M, A484E, and K512Y.co); (N-terminal biotinylation tag, N-terminal His10 tag, N-terminal Xa tag, T368F, E375Y, P477E, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, A176V, T368F, E375Y, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, T368F, E375Y, K422R, and K512Y); (N-terminal biotinylation tag, N-terminal His10 tag, N62D, E375Y, P477Q, A484E, and K512Y.co); (N-terminal biotinylation tag, N-terminal His10 tag, N-terminal Xa tag, 193F, 368F, E375Y, A484E, and K512Y); and (N-terminal biotinylation tag, N-terminal His10 tag, Y148A, E375Y, A484E, and K512Y.co).

[0014] Polymerases that include 193, 5215, E420, P477, D66R, K135R, K138R, L253T, Y369G, Y369L, L384M, K422A, I504R, E508K, E508R and/or D510K mutations optionally further include mutations at one or more of L253, T368, E375, A484 or K512. A polymerase that includes an 193 mutation optionally includes a mutation selected from 193F and 193Y. Polymerases that include an S215 mutation optionally include an S215D mutation. A polymerase that includes an E420 mutation can include an E420M mutation. When the polymerase includes a P477 mutation, the polymerase optionally includes a mutation selected from P477E and P477Q. When the polymerase includes a T368 mutation, the polymerase can include E375Y and K512Y mutations. In a related aspect, a polymerase that includes a T368 mutation can include E375Y, A484E and K512Y mutations.

[0015] Optionally, polymerases of the invention can include one or more amino acid insertions and/or deletions. For example, the one or more insertions can comprise an insertion of at least one amino acid (e.g., one, two or more amino acids) between positions (e.g., residues) 507 and 508, between positions 511 and 512, between positions 512 and 513, or a combination of insertions thereof.

[0016] As noted above, the compositions of the invention optionally include a polymerase that further includes one or more exogenous or heterologous features at the C-terminal and/or N-terminal region of the polymerase. For example, the one or more exogenous or heterologous features can be a polyhistidine tag, a HIS-10 tag, a HIS-6 tag, an alanine tag, an Ala10 tag, an Ala16 tag, a biotinylation tag (e.g., a Btag or BtagV7), a GST tag, a BiTag, an S Tag, a SNAP-tag, an HA tag, a DSB (Sso7D) tag, a lysine tag, a NanoTag, a Cmyc tag, a tag or linker comprising the amino acids glycine and serine, a tag or linker comprising the amino acids glycine, serine, alanine and histidine, a tag or linker comprising the amino acids glycine, arginine, lysine, glutamine and proline, a plurality of polyhistidine tags, a plurality of HIS-10 tags, a plurality of HIS-6 tags, a plurality of alanine tags, a plurality of Ala10 tags, a plurality of Ala16 tags, a plurality of biotinylation tags, a plurality of GST tags, a plurality of BiTags, a plurality of S Tags, a plurality of SNAP-tags, a plurality of HA tags, a plurality of DSB (Sso7D) tags, a plurality of lysine tags, a plurality of NanoTags, a plurality of Cmyc tags, a plurality of tags or linkers comprising the amino acids glycine and serine, a plurality of tags or linkers comprising the amino acids glycine, serine, alanine and histidine, a plurality of tags or linkers comprising the amino acids glycine, arginine, lysine, glutamine and proline, biotin, avidin, one or more Factor Xa sites, one or more enterokinase sites, thrombin sites, antibodies or antibody domains, antibody fragments, antigens, receptors, receptor domains, receptor fragments, ligands, dyes, acceptors, quenchers, or combinations thereof.

[0017] The polymerase can include one or more exogenous or heterologous features at both the C-terminal and N-terminal regions of the polymerase, where such features at the C-terminal and N-terminal regions are optionally the same, e.g., a polyhistidine tag (e.g., a His10 tag) at both the C-terminal and N-terminal regions. Polymerases that include exogenous or heterologous features at both the C-terminal and N-terminal regions optionally include a Btag and a polyhistidine tag (e.g., a Btag at the N-terminal region and a polyhistidine tag (e.g., a His-10 tag) at the C-terminal region). Polymerases that include a B-Tag and a polyhistidine tag can further include a Factor Xa recognition site.

[0018] The compositions optionally include the nucleotide analogue. Example nucleotide analogues include those that include fluorophore and/or dye moieties. For example, the nucleotide analogue can be a labeled nucleotide, e.g., a base-, sugar- and/or phosphate-labeled nucleotide. The analogue can be a mono-deoxy or a dideoxy nucleotide analogue.

[0019] One example class of nucleotide analogues are phosphate nucleotide analogues, including mono-deoxy phosphate nucleotide analogues and/or dideoxy phosphate nucleotide analogues, which analogues are optionally labeled on a phosphate moiety of the analogue (e.g., a delta phosphate). For example, the nucleotide analogue can be a labeled nucleotide analogue that includes 4 or more phosphate groups, e.g., where the nucleotide analogue is a tetraphosphate, a pentaphosphate, a hexaphosphate, or a septaphosphate analogue.

[0020] The compositions including a modified recombinant polymerase (e.g., any of the polymerases described above) can further include a phosphate-labeled nucleotide analog, a DNA template, where the polymerase can incorporate the nucleotide analog into a copy nucleic acid in response to the DNA template. These compositions can be present in a DNA sequencing system, e.g., a zero-mode waveguide. Optionally, the polymerase of the compositions can be immobilized on a surface. For example, the polymerase can be immobilized on the surface of a zero-mode waveguide in an active form.

[0021] Also provided by the present invention is a nucleic acid that encodes any of the polymerases described above, e.g., a nucleic acid encoding a recombinant DNA polymerase homologous to a Φ29 DNA polymerase, the polymerase including a mutation or combination of mutations selected from: an L253 mutation, where the polymerase further includes a mutation at one or more of T368, E375, A484, or K512; an E375 and K512 mutation, where the polymerase further includes a mutation at one or more of L253, T368 or A484; an 193 mutation; an S215 mutation; an E420 mutation; a P477 mutation; a D66R mutation; a K135R mutation; a K138R mutation; an L253T mutation; a Y369G mutation; a Y369L mutation; an L384M mutation; a K422A mutation; an I504R mutation; an E508K mutation; an E508R mutation; a D510K mutation; at least one mutation or combination of mutations selected from those listed in Tables 6, 9 and 10; and a T368 mutation, wherein the polymerase further includes a mutation at one or more of E375 or K512. Optionally, one or more codons of the nucleic acid is optimized for expression of the polymerase in a translation system, e.g., a cell (e.g., a prokaryotic or eukaryotic cell).

[0022] In a related aspect, the invention provides methods for sequencing a DNA template. Such methods include providing a reaction mixture that includes a DNA template, a replication initiation moiety that complexes with or is integral to the template, any of the recombinant DNA polymerases described above (e.g., a recombinant polymerase including a mutation or combination of mutations selected from: an L253 mutation, where the polymerase further includes a mutation at one or more of T368, E375, A484, or K512; an E375 and K512 mutation, where the polymerase further includes a mutation at one or more of L253, T368 or A484; an 193 mutation; an S215 mutation; an E420 mutation; a P477 mutation; a D66R mutation; a K135R mutation; a K138R mutation; an L253T mutation; a Y369G mutation; a Y369L mutation; an L384M mutation; a K422A mutation; an I504R mutation; an E508K mutation; an E508R mutation; a D510K mutation; at least one mutation or combination of mutations selected from those listed in Tables 6, 9 and 10; and a T368 mutation, where the polymerase further includes a mutation at one or more of E375 or K512), and one or more nucleotides and/or nucleotide analogs. The polymerase of the reaction mixture is generally capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction.

[0023] The methods of sequencing the DNA template include subjecting the reaction mixture to a polymerization reaction in which the modified recombinant polymerase replicates at least a portion of the template in a template-dependent manner by incorporating one or more nucleotides and/or nucleotide analogs into the resulting DNA. The methods also include identifying a time sequence of incorporation of the one or more nucleotides and/or nucleotide analogs into the resulting DNA. The nucleotide analogs used in the methods can comprise a first analog and a second analog (and optionally third, fourth, etc.), each of which comprise different fluorescent labels. The different fluorescent labels can optionally be distinguished from one another during the step in which a time sequence of incorporation is identified. Subjecting the reaction mixture to a polymerization reaction and identifying a time sequence of incorporation can optionally be performed in a zero mode waveguide.

[0024] In another aspect, the invention also provides methods of making a DNA that include providing a reaction mixture that includes a template, a replication initiating moiety that complexes with or is integral to the template, one or more nucleotides and/or nucleotide analogs, and a recombinant DNA polymerase, e.g., such as those described above, which can replicate at least a portion of the template using the moiety in a template-dependent polymerase reaction. The methods of making a DNA include reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA. Optionally, the reaction mixture can be reacted in a zero mode waveguide. The methods optionally include detecting the incorporation of at least one of the nucleotides and/or nucleotide analogs.

[0025] The present invention also provides methods of making the recombinant DNA polymerases described above (e.g., a recombinant polymerase including a mutation or combination of mutations selected from: an L253 mutation, where the polymerase further includes a mutation at one or more of T368, E375, A484, or K512; an E375 and K512 mutation, where the polymerase further includes a mutation at one or more of L253, T368 or A484; an I93 mutation; an S215 mutation; an E420 mutation; a P477 mutation; a D66R mutation; a K135R mutation; a K138R mutation; an L253T mutation; a Y369G mutation; a Y369L mutation; an L384M mutation; a K422A mutation; an I504R mutation; an E508K mutation; an E508R mutation; a D510K mutation; at least one mutation or combination of mutations selected from those listed in Tables 6, 9 and 10; and a T368 mutation, where the polymerase further includes a mutation at one or more of E375 or K512). Such methods include mutating the polymerase at a position relative to the wild-type Φ29 polymerase of SEQ ID NO:1 selected from: L253, where the polymerase is further mutated at one or more of T368, E375, A484 and K512; E375 and K512, where the polymerase is further mutated at one or more of L253, T368, or A484; 193; S215; E420; P477; D66, where the aspartic acid is mutated to arginine; K135, where the lysine is mutated to arginine; K138, where the lysine is mutated to arginine; L253, where the leucine is mutated to threonine; Y369, where the tyrosine is mutated to glycine; Y369, where the tyrosine is mutated to leucine; L384, where the leucine is mutated to methionine; K422, where the lysine is mutated to alanine; I504, where the isoleucine is mutated to arginine; E508, where glutamic acid is mutated to lysine; E508, where glutamic acid is mutated to arginine; D510, where the aspartic acid is mutated to lysine; a position or combination of positions selected from those listed in Tables 6, 9 and 10; and T368, where the polymerase is further mutated at one or more of E375 and K512.

[0026] In a related aspect, the invention provides compositions that include a modified recombinant DNA polymerase that exhibits a branching fraction that is less than about 25% for a phosphate-labeled nucleotide analog. The branching fraction is the proportion of cognate nucleotide (or nucleotide analog, e.g., A488dA4P) dissociation events from the polymerase active site to the total number of events, e.g., the sum of the incorporation events and dissociation events for the cognate nucleotide or analog. The polymerase of these compositions also exhibits a branching fraction that is less than a branching fraction for the analog exhibited by a parental polymerase, e.g., a wild-type or previously available mutant (e.g., exonuclease deficient) polymerase. The modified recombinant polymerase can be a modified recombinant Φ29-type DNA polymerase that comprises at least one mutation relative to the parental polymerase other than a T368D, T368E, or T368G substitution, where the numbering for the amino acid positions of the mutated residues is relative to that of the wild-type Φ29 polymerase. The compositions comprising a modified recombinant polymerase that exhibits a reduced branching fraction phenotype can optionally include the phosphate-labeled nucleotide analog, which can optionally comprise a fluorophore. The nucleotide analog of the compositions can optionally comprise from 3-7 phosphate groups.

[0027] The modified recombinant polymerase of the compositions described above can optionally exhibit a branching fraction of less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1% or even less than about 0.1% of the total interactions, e.g., association events and dissociation events, of the phosphate-labeled nucleotide analog, e.g., A488dA4P, with the nucleotide binding site of the polymerase. In comparison, a wild type Φ29 polymerase exhibits a branching fraction of about ≧40% for, e.g., a gamma-linked A488dA4P nucleotide analog, wherein ≧40% of the total events with a cognate gamma-linked A488dA4P nucleotide analog in the polymerase binding pocket are dissociation events. Alternately or additionally, the recombinant polymerases herein can exhibit a branching fraction that is less than 0.5× as high as a wild type parental polymerase (e.g., a wild type Φ29). Preferably, the branching fraction is about 0.25× as high as the parental polymerase or less, e.g., about 0.15× as high or less.

[0028] Optionally, the polymerase can exhibit a K_m for the phosphate-labeled nucleotide analog that is less than a K_m observed for a wild-type polymerase. This is particularly useful in applications in which the polymerase incorporates the analog, e.g., during SMS. For example, the modified recombinant polymerase can exhibit a Km for the phosphate-labeled nucleotide analog that is less than 75%, less than 50%, or less than 25% that of wild-type or parental polymerase such as a wild type Φ29). In one specific class of examples, the polymerases of the invention have a K_m of about 10 μM or less for a non-natural nucleotide analog such as a phosphate-labeled analog.

[0029] The modified recombinant polymerase of the compositions described above can optionally be a modified recombinant Φ29-type DNA polymerase. Optionally, the polymerase can be a modified recombinant Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, P7E, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase. The polymerase can optionally comprise at least one amino acid substitution or combination of substitutions including: an amino acid substitution at position 153; an amino acid substitution at position 191; an amino acid substitution at position 388; an amino acid substitution at position 422; an amino acid substitution at position 128; an amino acid substitution at position 253; an amino acid substitution at position 504; an amino acid substitution at position 143; an amino acid substitution at position 183; an amino acid substitution at position 236; an amino acid substitution at position 363; P153L; G191A; T368F; T368P; T368S; T368V; T368N; T368A; T373N; T373V; T373C; I378V; I378F; K379S; K379A; S388A; S388T; K422R; F128M; F128V; I504V; K143D; K512R; Q183S; R236N; L253A; F363Y; L253A, F363Y, and L480M; T368F, K379S, E375Y, and K512Y; T368F and K379S; T368G and K379S; T368F and T373A; E375Y, K512Y and K379S; E375Y, K512Y and T368F; T368F and V514K; T368F and K379T; S388A and P153L; E375Y, K512Y and T368G; T368G and T373A; E375W and T368G; I378K and K379S; T368F and I378K; T368G and I378K; T368G and V514K; E375W and K379T; T373A and K379S; E375W and T373A; E375Y, K512Y and T373A; E375W and I378K; E375Y, K512Y and I378K; T373A and V514K; T373A and I378K; E375Y, K512Y and K379T; I378K and V514K; E375W and V514K; T368G and K379T; and/or E375Y, K512Y and V514K, where the numbering for the amino acid positions of the mutated residues is relative to that of the wild-type Φ29 polymerase. The modified recombinant polymerase of the compositions described above can optionally further comprise an amino acid substitution selected from the group consisting of T368D, T368E, T368G, E375Y, E375W, K512Y, K512F, K512W, K512L, K512I, K512V, and K512H, or can include one of these substitutions at an indicated position (e.g., K512F where K512Y is listed). Optionally, the polymerase can comprise a mutation that inhibits exonuclease activity of the polymerase, such as an N62D, D12A, D66A, or T15I mutation.

[0030] The compositions comprising a modified recombinant polymerase that exhibits a reduced branching fraction phenotype, e.g., a branching fraction phenotype described above, can include a phosphate-labeled nucleotide analog, a DNA template, and a modified recombinant DNA polymerase, e.g., any of the polymerases described above, that can incorporate the nucleotide analog into a copy nucleic acid in response to the DNA template. These compositions can be present in a DNA sequencing system, e.g., a zero-mode waveguide. Optionally, the polymerase of the compositions can be immobilized on a surface.

[0031] In a related aspect, the invention provides methods of sequencing a DNA template. The methods include providing a reaction mixture that includes the DNA template, a replication initiating moiety that complexes with or is integral to the template, one or more nucleotides and/or nucleotide analogs, and a modified recombinant DNA polymerase, e.g., any of those described above. The polymerase of the reaction mixture is generally capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction.

[0032] The methods of sequencing the DNA template include subjecting the reaction mixture to a polymerization reaction in which the modified recombinant polymerase replicates at least a portion of the template in a template-dependent manner by incorporating one or more nucleotides and/or nucleotide analogs into the resulting DNA. The methods also include identifying a time sequence of incorporation of the one or more nucleotides and/or nucleotide analogs into the resulting DNA. The nucleotide analogs used in the methods can comprise a first analog and a second analog (and optionally third, fourth, etc.), each of which comprise different fluorescent labels. The different fluorescent labels can optionally be distinguished from one another during the step in which a time sequence of incorporation is identified. Subjecting the reaction mixture to a polymerization reaction and identifying a time sequence of incorporation can optionally be performed in a zero mode waveguide.

[0033] In another aspect, the invention also provides methods of making a DNA that include providing a reaction mixture that comprises a template, a replication initiating moiety that complexes with or is integral to the template, one or more nucleotides and/or nucleotide analogs, and a modified recombinant DNA polymerase, e.g., such as those described above, which can replicate at least a portion of the template using the moiety in a template-dependent polymerase reaction. The methods of making a DNA include reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA. Optionally, the reaction mixture can be reacted in a zero mode waveguide. Optionally, the method includes detecting the incorporation of at least one of the nucleotides and/or nucleotide analogs.

[0034] The invention also features compositions that include a modified recombinant Φ29-type DNA polymerase that comprises at least one amino acid substitution or combination of substitutions selected from: an amino acid substitution at position 153; an amino acid substitution at position 191; an amino acid substitution at position 388; an amino acid substitution at position 422; an amino acid substitution at position 128; an amino acid substitution at position 253; an amino acid substitution at position 504; an amino acid substitution at position 143; an amino acid substitution at position 183; an amino acid substitution at position 236; an amino acid substitution at position 363; P153L; G191A; T368F; T368P; T368S; T368V; T368N; T368A; T373N; T373V; T373C; I378V; I378F; K379S; K379A; S388A; S388T; K422R; F128M; F128V; I504V; K143D; K512R; Q183S; R236N; L253A; F363Y; L253A, F363Y, and L480M; T368F, K379S, E375Y, and K512Y; T368F and K379S; T368G and K379S; T368F and T373A; E375Y, K512Y and K379S; E375Y, K512Y and T368F; T368F and V514K; T368F and K379T; S388A and P153L; E375Y, K512Y and T368G; T368G and T373A; E375W and T368G; I378K and K379S; T368F and I378K; T368G and I378K; T368G and V514K; E375W and K379T; T373A and K379S; E375W and T373A; E375Y, K512Y and T373A; E375W and I378K; E375Y, K512Y and I378K; T373A and V514K; T373A and I378K; E375Y, K512Y and K379T; I378K and V514K; E375W and V514K; T368G and K379T; and E375Y, K512Y and V514K, where the numbering for the amino acid positions of the mutated residues is relative to that of the wild-type Φ29 polymerase. The modified recombinant polymerase of the compositions described above can optionally include one or more of amino acid substitution T368D, T368E, T368G, E375Y, E375W, K512Y, K512F, K512W, K512L, K512I, K512V, or K512H, or can include one of these substitutions at an indicated position (e.g., K512F where K512Y is listed). Optionally, the polymerase of the compositions can be exonuclease deficient. Essentially all of the features noted above apply to these embodiments as well, as relevant, e.g., with respect to inclusion of analogs in the composition, immobilization of the polymerase on a surface, use of the polymerase in sequencing or making DNA, and the like.

[0035] In a related aspect, the invention provides methods of making a modified recombinant DNA polymerase that include structurally modeling a parental DNA polymerase, e.g., a Φ29-type DNA polymerase, identifying one or more complex stability or nucleotide interaction feature affecting complex stability or nucleotide access or binding in the active site or a complementarity feature for a nucleotide analog at the active site, and mutating the parental DNA polymerase to include or remove these features. For example, the polymerase can be mutated to improve steric access of the nucleotide analog to the active site or to improve charge-charge or hydrophobic interactions between the nucleotide analog and the polymerase. The methods also include determining whether the resulting modified recombinant polymerase displays a decreased branching fraction for a nucleotide or nucleotide analog as compared to the parental polymerase.

[0036] Determining whether the resulting modified recombinant polymerase displays a decreased branching fraction for a nucleotide or nucleotide analog as compared to the parental polymerase can optionally include determining whether the modified recombinant polymerase displays a decreased branching fraction for a phosphate-labeled nucleotide analog, e.g., an analog including 3-7 phosphate groups such as those described herein. The methods optionally include making a library of modified recombinant polymerases, a plurality of which comprise one or more mutations at the one or more positions. Optionally, the methods include screening the library to identify at least one member exhibiting a decreased branching fraction.

[0037] Another aspect of the invention provides compositions that include a modified recombinant DNA polymerase that comprises at least one mutation relative to a parental polymerase that increases, or is predicted to increase, the stability of a closed complex comprising the recombinant DNA polymerase and a DNA (relative to a closed complex including the parental polymerase and DNA). A mutation that stabilizes the closed complex can optionally stabilize an interaction between the thumb, TPR2, and/or exonuclease domains.

[0038] The stability of the closed complex can optionally be measured by determining a dissociation rate or dissociation rate constant (k_off) for dissociation of the polymerase from the DNA. The dissociation rate constant (k_off) for dissociation of the polymerase from the DNA is optionally measured in the presence of an excess of a competitor of the DNA, e.g., heparin or a population of DNA molecules. Optionally, dissociation can be indirectly measured by measuring an activity of the DNA polymerase over time. Optionally, the stability of the closed complex can be determined by measuring K_d for the complex. The increased stability of the complex can optionally include an improvement of at least about 30%.

[0039] The recombinant DNA polymerase that can form a more stable closed complex with a DNA can optionally exhibit increased processivity as compared to the parental polymerase. Processivity can be defined as (k_ext)/(k_ext+k_off), wherein k_ext is the extension rate constant and k_off is the rate constant for polymerase dissociation from the DNA. Optionally, the mutation decreases k_off. Processivity of the modified recombinant polymerase can optionally be at least twice that of the parental polymerase. The DNA of the compositions can optionally comprise a template strand and a primer.

[0040] The recombinant DNA polymerase that includes at least one mutation relative to a parental polymerase that increases, or is predicted to increase, the stability of a closed conformation of a complex comprising the recombinant DNA polymerase and a DNA can optionally be homologous to a Φ29 DNA polymerase, a Taq polymerase, an exonuclease deficient Taq polymerase, a DNA Pol I polymerase, a T7 polymerase, a T5 Polymerase, an RB69 polymerase, a T5 polymerase or a polymerase corresponding to a Klenow fragment of a DNA Pol I polymerase. Optionally the polymerase can be homologous to one or more polymerases, including a wild-type Φ29 DNA polymerase, an exonuclease deficient Φ29 DNA polymerase, a B103 DNA polymerase, a GA-1 DNA polymerase, a PZA DNA polymerase, a Φ15 DNA polymerase, a BS32 DNA polymerase, a M2Y DNA polymerase, an Nf DNA polymerase, a G1 DNA polymerase, a Cp-1 DNA polymerase, a PRD1 DNA polymerase, a PZE DNA polymerase, an SF5 DNA polymerase, a Cp-5 DNA polymerase, a Cp-7 DNA polymerase, a PR4DNA polymerase, a PR5DNA polymerase, a PR722DNA polymerase, or an L17 DNA polymerase. Optionally, the modified recombinant polymerase can be a modified recombinant Φ29-type DNA polymerase, which modified recombinant polymerase comprises at least one mutation relative to the parental polymerase at one or more positions selected from the group consisting of position 68-position 76, position 92, position 405-position 413, and position 560-position 564, where the numbering for the amino acid positions of the mutated residues is relative to that of the wild-type Φ29 polymerase. For example, the polymerase optionally comprises at least one amino acid substitution or combination of substitutions from T92F, T92I, G410D, T92I and G410D, N72A, N72I, N72F, and N72S. Essentially all of the features noted above apply to these embodiments as well, as relevant, e.g., with respect to inclusion of analogs in the composition, immobilization of the polymerase on a surface, use of the polymerase in sequencing or making DNA, and the like.

[0041] A related aspect of the invention provides compositions comprising a modified recombinant Φ29-type DNA polymerase that comprises one or more amino acid substitutions relative to a parental polymerase at one or more positions selected from the group consisting of position 68-position 76, position 92, position 405-position 413, and position 560-position 564, where the numbering for the amino acid positions of the mutated residues is relative to that of the wild-type Φ29 polymerase. The modified recombinant polymerase of the compositions exhibits increased stability of a closed complex comprising the recombinant DNA polymerase and a DNA as compared to the parental polymerase.

[0042] The recombinant and parental polymerase of the compositions can optionally be homologous to a Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase. Optionally, the modified recombinant polymerase is a modified recombinant Φ29 polymerase that comprises at least one amino acid substitution or combination of substitutions that include T92F, T92I, G410D, T92I and G410D, N72A, N72I, N72F, and N72S.

[0043] The compositions that comprise a modified recombinant polymerase that can form a more stable closed complex with a DNA can optionally include a nucleotide analog. The analog can optionally comprise a fluorophore, a phosphate-labeled nucleotide analog, and/or a labeled nucleotide analog having from 3-7 phosphate groups. Optionally, the compositions can include a nucleotide analog and a DNA template, and the modified recombinant polymerase of the composition can incorporate the nucleotide analog into a copy nucleic acid in response to the DNA template. The composition can optionally be present in a DNA sequencing system, e.g., a zero-mode waveguide. The one or more amino acid substitutions of the modified recombinant polymerase in the compositions can optionally increase, or be predicted to increase, the stability of a closed conformation of the modified recombinant polymerase relative to the parental polymerase. Optionally, the modified recombinant polymerase can be immobilized on a surface. Essentially all of the features noted above apply to these embodiments as well, as relevant.

[0044] In a related aspect, the invention provides methods of sequencing a DNA template that include providing a reaction mixture. The reaction mixture includes the DNA template, a replication initiating moiety that complexes with or is integral to the template, one or more nucleotides and/or nucleotide analogs, and a modified recombinant Φ29-type DNA polymerase that comprises one or more amino acid substitutions relative to a parental polymerase at one or more positions selected from the group consisting of position 68-position 76, position 92, position 405-position 413, and position 560-position 564, where the numbering for the amino acid positions of the mutated residues is relative to that of the wild-type Φ29 polymerase. The polymerase of the reaction mixture can be capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction.

[0045] The methods of sequencing a DNA template include subjecting the reaction mixture to a polymerization reaction in which the modified recombinant polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA. The methods also include identifying a time sequence of incorporation of the one or more nucleotides and/or nucleotide analogs into the resulting DNA.

[0046] The nucleotide analogs used in the methods can comprise a first analog and a second analog (and optionally third, fourth, etc.), each of which comprise different fluorescent labels. The different fluorescent labels can optionally be distinguished from one another during the step in which a time sequence of incorporation is identified. Optionally, subjecting the reaction mixture to a polymerization reaction and identifying a time sequence of incorporation can be performed in a zero mode waveguide.

[0047] In another related aspect, the invention provides methods of making a DNA that include providing a reaction mixture. The reaction mixture includes a template, a replication initiating moiety that complexes with or is integral to the template, one or more nucleotides and/or nucleotide analogs, and a modified recombinant Φ29-type DNA polymerase that comprises one or more amino acid substitutions relative to a parental polymerase at one or more positions selected from the group consisting of position 68-position 76, position 92, position 405-position 413, and position 560-position 564, where the numbering for the amino acid positions of the mutated residues is relative to that of the wild-type Φ29 polymerase. The polymerase in the reaction mixture can be capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction.

[0048] The methods include reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA. The reaction mixture used in the methods can optionally be reacted in a zero mode waveguide. The method can optionally include detecting incorporation of at least one of the nucleotides and/or nucleotide analogs.

[0049] In another aspect, the invention also provides methods of making a modified recombinant DNA polymerase. These methods include structurally modeling a parental DNA polymerase, identifying one or more amino acid residue positions in the parental DNA polymerase as targets for mutation, and mutating the parental polymerase at the one or more positions to introduce at least one intramolecular interaction predicted to stabilize the closed complex of the polymerase or to remove at least one intramolecular interaction predicted to destabilize the closed complex. The methods include determining whether the resulting modified recombinant polymerase displays an increased processivity, decreased k_off, or decreased K_d, as compared to the parental polymerase.

[0050] The methods can optionally include making a library of modified recombinant polymerases that comprise one or more mutations at the one or more positions. Optionally, the methods can include screening the library to identify at least one member exhibiting increased processivity, decreased k_off, or decreased K_d. The parental DNA polymerase can optionally be a Φ29-type DNA polymerase, and the amino acid positions identified in the parental DNA polymerase as targets for mutation can comprise at least one residue selected from position 68-position 76, position 92, position 405-position 413, and position 560-position 564, where the numbering for the amino acid positions of the mutated residues is relative to that of the wild-type Φ29 polymerase.

[0051] In another aspect, the invention provides a composition that includes a modified recombinant DNA polymerase that comprises one or more mutations relative to a parental polymerase and that exhibits a first rate constant for a first step in its catalytic cycle (generally, the forward rate constant for the first step) that is less than a first rate constant for the first step exhibited by the parental polymerase (e.g., a wild-type or previously available mutant polymerase, e.g., an exonuclease-deficient polymerase). For example, the first rate constant exhibited by the modified recombinant polymerase can be less than 0.25 times the first rate constant exhibited by the parental polymerase, e.g., less than about 0.1 times.

[0052] As discussed in greater detail herein, decreasing the rate of certain steps within the catalytic cycle can improve accuracy in single molecule sequencing techniques, for example, by reducing the number of times that incorporation of a labeled nucleotide with release of the label is too fast to be detected. Slowing the rate of a step which is not normally rate limiting in the catalytic cycle can be particularly useful in this regard. Manipulating the rate of the first step so that it is comparable to that of another step, particularly one that is already relatively slow, is also particularly useful in this regard. Thus, the modified recombinant polymerase optionally exhibits a second rate constant for a second step in its catalytic cycle (generally, the forward rate constant for the second step), where the second rate constant is between 0.1 and 10 times the first rate constant, preferably between 0.2 and 5 times the first rate constant, and more preferably approximately equal to the first rate constant. It will be understood that in this context, the terms first step and second step are merely used for convenience in referring to two different steps and do not imply any particular order of occurrence (that is, the first step can precede or follow the second and need not be the initial event in the catalytic pathway).

[0053] The second step is optionally rate limiting in the catalytic cycle of the parental polymerase (and optionally also of the modified polymerase). More generally, however, the first and/or second step can be limiting in a portion of the cycle, not necessarily the entire cycle. For example, the first and second steps can be limiting in the bright portion of the cycle where a label from a labeled analog is retained in a complex with the polymerase, or in the dark portion of the cycle where the label is not associated with the polymerase. As one example, the second step can involve incorporation of a bound nucleotide or nucleotide analog, e.g., an analog having from 3-7 phosphate groups, e.g., with a terminal label.

[0054] Any of a number of steps can be slowed using the techniques of the instant invention. In one aspect, product release, particularly polyphosphate product release, is slowed (i.e., the first step involves release of a polyphosphate product). Optionally, the first rate constant exhibited by the modified recombinant polymerase is less than about 100/second, e.g., less than about 75/second or even less than about 50/second. In one exemplary embodiment, the second step involves incorporation of a bound nucleotide or nucleotide analog, the first step involves release of a polyphosphate product, and the second rate constant exhibited by the modified recombinant polymerase is between 0.1 and 10 times (e.g., between 0.2 and 5 times or between 0.2 and 1 times) the first rate constant exhibited by the modified recombinant polymerase.

[0055] It will be evident that the rate of the second step can also be manipulated, e.g., to balance the rates of the first and second steps. Thus, the second rate constant exhibited by the modified recombinant polymerase is optionally smaller (faster) than the second rate constant exhibited by the parental polymerase for the second step.

[0056] The modified recombinant polymerase can be a modified recombinant Φ29-type DNA polymerase, for example, a modified recombinant Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZb, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase. The polymerase optionally comprises at least one amino acid substitution or combination of substitutions selected from the group consisting of: an amino acid substitution at position 484; an amino acid substitution at position 198; an amino acid substitution at position 381; A484E; A484Y; N387L; T372Q; T372Y; T372Y and K478Y; K478Y; I370W; F198W; L381A; T368F; A484E, E375Y, K512Y, and T368F; A484Y, E375Y, K512Y, and T368F; N387L, E375Y, K512Y, and T368F; T372Q, E375Y, K512Y, and T368F; T372L, E375Y, K512Y, and T368F; T372Y, K478Y, E375Y, K512Y, and T368F; I370W, E375Y, K512Y, and T368F; F198W, E375Y, K512Y, and T368F; L381A, E375Y, K512Y, and T368F; and E375Y, K512Y, and T368F; wherein numbering of positions is relative to wild-type Φ29 polymerase. Additional exemplary mutations are described herein. The polymerase can include a mutation that inhibits exonuclease activity of the polymerase, e.g., an N62D, D12A, D66A, or T15I substitution.

[0057] The composition optionally also includes one or more nucleotide analog, for example, a phosphate-labeled nucleotide analog, e.g., one comprising a fluorophore and/or having from 3-7 phosphate groups. Similarly, the composition can include a phosphate-labeled nucleotide analog and a DNA template, wherein the modified recombinant polymerase incorporates the nucleotide analog into a copy nucleic acid in response to the DNA template. The composition can be present in a DNA sequencing system, e.g., a zero mode waveguide. Optionally, the polymerase is immobilized on a surface.

[0058] In a related aspect, the invention provides methods of sequencing a DNA template. In the methods, a reaction mixture that includes the DNA template, a replication initiating moiety that complexes with or is integral to the template, one or more nucleotides and/or nucleotide analogs, and a modified recombinant DNA polymerase, e.g., any of those described above, is provided. The polymerase is generally capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction.

[0059] The reaction mixture is subjected to a polymerization reaction in which the modified recombinant polymerase replicates at least a portion of the template in a template-dependent manner by incorporating one or more nucleotides and/or nucleotide analogs into the resulting DNA. A time sequence of incorporation of the one or more nucleotides and/or nucleotide analogs into the resulting DNA is identified. The nucleotide analogs used in the methods can comprise a first analog and a second analog (and optionally third, fourth, etc.), each of which comprise different fluorescent labels. The different fluorescent labels are optionally distinguished from one another during the step in which a time sequence of incorporation is identified. The polymerization reaction and the identification step are optionally performed in a zero mode waveguide.

[0060] In another aspect, the invention also provides methods of making a DNA that include providing a reaction mixture that comprises a template, a replication initiating moiety that complexes with or is integral to the template, one or more nucleotides and/or nucleotide analogs, and a modified recombinant DNA polymerase, e.g., such as those described above, which can replicate at least a portion of the template using the moiety in a template-dependent polymerase reaction. The methods of making a DNA include reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA. The reaction mixture is optionally reacted in a zero mode waveguide. Optionally, the method includes detecting the incorporation of at least one of the nucleotides and/or nucleotide analogs.

[0061] One general class of embodiments provides a composition comprising a modified recombinant DNA polymerase, e.g., a modified recombinant Φ29-type DNA polymerase, that exhibits a rate constant for release of a polyphosphate product that is less than about 50/second. Essentially all of the features noted above apply to these embodiments as well, as relevant, e.g., with respect to inclusion of analogs in the composition, immobilization of the polymerase on a surface, use of the polymerase in sequencing or making DNA, and the like. The modified polymerase can include mutations detailed herein, for example, one or more amino acid substitution or combination of substitutions selected from: an amino acid substitution at position 484; an amino acid substitution at position 198; an amino acid substitution at position 381; A484E; A484Y; N387L; T372Q; T372Y; T372Y and K478Y; K478Y; I370W; F198W; L381A; T368F; A484E, E375Y, K512Y, and T368F; A484Y, E375Y, K512Y, and T368F; N387L, E375Y, K512Y, and T368F; T372Q, E375Y, K512Y, and T368F; T372L, E375Y, K512Y, and T368F; T372Y, K478Y, E375Y, K512Y, and T368F; I370W, E375Y, K512Y, and T368F; F198W, E375Y, K512Y, and T368F; L381A, E375Y, K512Y, and T368F; and E375Y, K512Y, and T368F.

[0062] Another general class of embodiments provides a composition comprising a modified recombinant polymerase, e.g., a modified recombinant Φ29-type DNA polymerase, that comprises one or more mutations relative to a parental polymerase at one or more positions selected from the group consisting of a) positions that form a binding site for a metal ion that interacts with an epsilon and/or digamma phosphate of a bound nucleotide analog having five or more phosphate groups; b) positions 372-397 and 507-514; c) positions that form a binding site for a terminal fluorophore on a phosphate-labeled nucleotide analog; d) positions at an intramolecular interface in a closed conformation of a ternary complex comprising the polymerase, a DNA, and a nucleotide or nucleotide analog; e) positions that form a binding site for a polyphosphate group of a bound nucleotide or nucleotide analog; f) positions that interact with the base of a bound nucleotide or nucleotide analog; and g) positions that interact with a bound DNA; wherein numbering of positions is relative to wild-type Φ29 polymerase. Typically, the one or more mutations comprise at least one mutation other than a 514Y, 514W, 514F, 514I, 514K, 259S, 370V, 370K, 372D, 372E, 372R, 372K, 372N, 372L, 387A, 387D, 478D, 478E, 478R, 480K, 480M, 480R, 371Q, 379E, 379T, 486D, 486A, 188A, 188S, 254F, 254V, 254A, 390F, or 390A substitution.

[0063] The recombinant and parental polymerase are optionally homologous to a Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.

[0064] The one or more mutations can include at least one amino acid substitution, for example, at least one amino acid substitution at least one residue selected from the group consisting of positions 484, 249, 179, 198, 211, 255, 259, 360, 363, 365, 370, 372, 378, 381, 383, 387, 389, 393, 433, 478, 480, 514, 251, 371, 379, 380, 383, 458, 486, 101, 188, 189, 303, 313, 395, 414, 497, 500, 531, 532, 534, 558, 570, 572, 574, 64, 305, 392, 402, 422, 496, 529, 538, 555, 575, 254, and 390. Exemplary substitutions and combinations thereof include, but are not limited to, A484E; A484Y; N387L; T372Q; T372Y; T372Y and K478Y; K478Y; I370W; F198W; L381A; T368F; A484E, E375Y, K512Y, and T368F; A484Y, E375Y, K512Y, and T368F; N387L, E375Y, K512Y, and T368F; T372Q, E375Y, K512Y, and T368F; T372L, E375Y, K512Y, and T368F; T372Y, K478Y, E375Y, K512Y, and T368F; I370W, E375Y, K512Y, and T368F; F198W, E375Y, K512Y, and T368F; L381A, E375Y, K512Y, and T368F; and E375Y, K512Y, and T368F. Exemplary mutations also include insertions and deletions. Thus, for example, the one or more mutations can comprise an insertion of at least one amino acid within residues 372-397 and/or 507-514. Other exemplary mutations are described herein.

[0065] Essentially all of the features noted above apply to these embodiments as well, as relevant, e.g., with respect to inclusion of analogs in the composition, immobilization of the polymerase on a surface, use of the polymerase in sequencing or making DNA, and the like.

[0066] A related aspect of the invention provides methods of making a modified recombinant DNA polymerase that include structurally modeling a parental DNA polymerase, identifying one or more amino acid residue positions in the parental DNA polymerase as targets for mutation, mutating the parental polymerase at the one or more positions, and determining whether the resulting modified recombinant polymerase exhibits a first rate constant for a first step in its catalytic cycle that is less than a first rate constant for the first step exhibited by the parental polymerase. The methods optionally also include determining whether the resulting modified polymerase exhibits a second rate constant for a second step where the second rate constant is between 0.1 and 10 times the first rate constant.

[0067] A number of strategies for producing such polymerases are described herein. For example, the parental DNA polymerase and a bound nucleotide analog having five or more phosphate group can be modeled, and one or more amino acid residues of the parental DNA polymerase that form a binding site for a metal ion that interacts with an epsilon and/or digamma phosphate of the analog can be identified as the targets for mutation. In another example, the parental polymerase is mutated at the one or more positions to introduce at least one intramolecular interaction predicted to stabilize a closed conformation of a ternary complex comprising the polymerase, a DNA, and a nucleotide or nucleotide analog or to remove at least one intramolecular interaction predicted to destabilize the closed conformation.

[0068] In another example, the parental polymerase is mutated at the one or more positions to increase intermolecular interaction between the polymerase and the base of an incoming nucleotide or nucleotide analog. In yet another example, the parental polymerase is mutated at the one or more positions to increase intermolecular interaction between the polymerase and a terminal fluorophore on a nucleotide analog. In an additional example, the parental DNA polymerase is modeled with a bound nucleotide analog having four or more phosphate groups, and the parental polymerase is mutated at the one or more positions to alter isomerization of the phosphate groups. In a related example, the parental DNA polymerase is modeled with a bound polyphosphate, and the parental polymerase is mutated at the one or more positions to alter isomerization of the polyphosphate. In another example, the parental DNA polymerase and a DNA are modeled, and one or more amino acid residues of the parental DNA polymerase that interact with the DNA and/or that are within 4 Å of the DNA are identified as targets for mutation.

[0069] The methods can be applied to any of the large number of polymerases available in the art, for example, a Φ29-type DNA polymerase. In one exemplary class of embodiments, the parental polymerase is a Φ29-type DNA polymerase, and the one or more positions identified in the parental DNA polymerase as targets for mutation comprise at least one residue selected from the group consisting of position 484, 249, 179, 198, 211, 255, 259, 360, 363, 365, 370, 372, 378, 381, 383, 387, 389, 393, 433, 478, 480, 514, 372-397, 507-514, 251, 371, 379, 380, 383, 458, 486, 101, 188, 189, 303, 313, 395, 414, 497, 500, 531, 532, 534, 558, 570, 572, 574, 64, 305, 392, 402, 422, 496, 529, 538, 555, 575, 254, and 390, wherein numbering of positions is relative to wild-type Φ29 DNA polymerase.

[0070] The methods optionally include making a library of modified recombinant polymerases, a plurality of which comprise one or more mutations at the one or more positions. Optionally, the methods include screening the library to identify at least one member exhibiting the decreased first rate constant. Similarly, the library is optionally screened to identify at least one member exhibiting comparable first and second rate constants, as described herein.

[0071] Essentially all of the features noted for the compositions above apply to these embodiments as well, as relevant, e.g., with respect to type of first and/or second steps, balance of the first and second rate constants, type of parental polymerase, and the like.

[0072] The present invention also features kits that include the polymerases of the invention, optionally with additional useful reagents such as one or more nucleotide analogs, e.g., for sequencing, nucleic acid amplification, or the like. Such kits can include the polymerase of the invention packaged in a fashion to enable use of the polymerase, a set of different nucleotide analogs of the invention, e.g., those that are analogous to A, T, G, and C, e.g., where one or more of the analogs comprise a detectable moiety to permit identification in the presence of the analogs. Depending upon the desired application, the kits of the invention optionally include additional reagents, such as natural nucleotides, a control template, and other reagents, such as buffer solutions and/or salt solutions, including, e.g., divalent metal ions such as Ca⁺⁺, Mg⁺⁺, Mn⁺⁺ and/or Fe⁺⁺, and standard solutions, e.g., dye standards for detector calibration. Such kits also typically include instructions for use of the polymerases and other reagents in accordance with the desired application methods, e.g., nucleic acid sequencing, amplification and the like. DNA sequencing systems that include any of the modified polymerases described herein are also a feature of the invention. Such systems can optionally include detectors, excitation light sources, array readers, and the like.

[0073] Those of skill in the art will appreciate that that the methods provided by the invention for modifying a DNA polymerase, e.g., to decrease branching fraction formation during polymerization, increase closed polymerase/DNA complex stability, and/or slow one or more steps in the catalytic cycle, can be used alone or in combination. Accordingly, all of the various mutations described herein can appear singly or in combination in a modified recombinant polymerase.

[0107] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0108] As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a protein” includes a plurality of proteins; reference to “a cell” includes mixtures of cells, and the like.

[0109] The term “about” as used herein indicates the value of a given quantity varies by +/−10% of the value, or optionally +/−5% of the value, or in some embodiments, by +/−1% of the value so described.

[0110] The term “nucleic acid” or “polynucleotide” encompasses any physical string of monomer units that can be corresponded to a string of nucleotides, including a polymer of nucleotides (e.g., a typical DNA or RNA polymer), PNAs, modified oligonucleotides (e.g., oligonucleotides comprising nucleotides that are not typical to biological RNA or DNA, such as 2′-O-methylated oligonucleotides), and the like. A nucleic acid can be e.g., single-stranded or double-stranded. Unless otherwise indicated, a particular nucleic acid sequence of this invention encompasses complementary sequences, in addition to the sequence explicitly indicated.

[0111] A “polypeptide” is a polymer comprising two or more amino acid residues (e.g., a peptide or a protein). The polymer can additionally comprise non-amino acid elements such as labels, quenchers, blocking groups, or the like and can optionally comprise modifications such as glycosylation or the like. The amino acid residues of the polypeptide can be natural or non-natural and can be unsubstituted, unmodified, substituted or modified.

[0112] An “amino acid sequence” is a polymer of amino acid residues (a protein, polypeptide, etc.) or a character string representing an amino acid polymer, depending on context.

[0113] A “polynucleotide sequence” or “nucleotide sequence” is a polymer of nucleotides (an oligonucleotide, a DNA, a nucleic acid, etc.) or a character string representing a nucleotide polymer, depending on context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence (e.g., the complementary nucleic acid) can be determined.

[0114] Numbering of a given amino acid or nucleotide polymer “corresponds to numbering of” or is “relative to” a selected amino acid polymer or nucleic acid when the position of any given polymer component (amino acid residue, incorporated nucleotide, etc.) is designated by reference to the same residue position in the selected amino acid or nucleotide, rather than by the actual position of the component in the given polymer. Correspondence of positions is typically determined by aligning the relevant amino acid or polynucleotide sequences.

[0115] The term “recombinant” indicates that the material (e.g., a nucleic acid or a protein) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. For example, a “recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other procedures, or by chemical or other mutagenesis; a “recombinant polypeptide” or “recombinant protein” is, e.g., a polypeptide or protein which is produced by expression of a recombinant nucleic acid.

[0116] A “Φ29-type DNA polymerase” (or “phi29-type DNA polymerase”) is a DNA polymerase from the Φ29 phage or from one of the related phages that, like Φ29, contain a terminal protein used in the initiation of DNA replication. Φ29-type DNA polymerases are homologous to the Φ29 DNA polymerase; examples include the B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, and L17 DNA polymerases, as well as chimeras thereof. A modified recombinant Φ29-type DNA polymerase includes one or more mutations relative to naturally-occurring wild-type Φ29-type DNA polymerases, for example, one or more mutations that increase closed complex stability, decrease branching fraction, and/or slow a catalytic step relative to a corresponding wild-type polymerase, and may include additional alterations or modifications over wild-type Φ29-type DNA polymerases, such as deletions, insertions, and/or fusions of additional peptide or protein sequences (e.g., for immobilizing the polymerase on a surface or otherwise tagging the polymerase enzyme).

[0117] A variety of additional terms are defined or otherwise characterized herein.

[0118] One aspect of the invention is generally directed to modified or engineered polymerases that are characterized by lowered frequency of branching events during polymerization reactions, increased stability of closed polymerase-DNA complexes, and/or decreased rates for steps in the polymerization cycle. Individually or in combination, these modifications can increase polymerase processivity and/or polymerase activity readout accuracy (e.g., increase sequence accuracy in single molecule sequencing reactions). Polymerases of the invention optionally include additional mutations that provide other desirable features, e.g., that eliminate exonuclease or proof reading activity of the relevant polymerase, increase residence time of nucleotide analogs at an active site of the polymerase, modify one or more kinetic feature of the polymerase, increase surface stability for polymerases bound to a surface, or the like.

[0119] Compositions that include modified recombinant DNA polymerases that include amino acid substitutions, insertions, deletions and/or heterologous or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing are a feature of the invention. Relative to a wild-type Φ29 DNA polymerase, these modifications can include any one of, or any combination of: an L253 mutation and a mutation at one or more of T368, E375, A484, or K512; an E375 and K512 mutation, and a mutation at one or more of L253, T368 or A484; an I93 mutation; an S215 mutation; an E420 mutation; a P477 mutation; a D66R mutation; a K135R mutation; a K138R mutation; an L253T mutation; a Y369G mutation; a Y369L mutation; an L384M mutation; a K422A mutation; an I504R mutation; an E508K mutation; an E508R mutation; a D510K mutation; or at least one mutation or combination of mutations selected from those listed in Tables 6, 9 and 10. The modified polymerases can exhibit desirable features described in detail hereinbelow, e.g., reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fractions, increased closed complex stability, enhanced metal ion coordination and/or reduced exonuclease activity, etc.

[0120] As noted, polymerases exhibiting a decreased branching fraction are a feature of the invention. “Branching” is a phenomenon that occurs during polymerization. During a polymerase kinetic cycle, sampling of each of four possible nucleotides (or analogs) occurs until a correct Watson-Crick pairing is generated (see, e.g., Hanzel et al. WO 2007/076057 POLYMERASES FOR NUCLEOTIDE ANALOGUE INCORPORATION for a description of the kinetic cycle of a polymerase; see also the section entitled “Polymerase Mediated Synthesis” hereinbelow). However, chemical linkages between a sampled nucleotide and a 3′ hydroxyl group of a preceding base can fail to occur for a correctly paired nucleotide, due to release of the correctly paired base from the active site. Such failures to physically incorporate the correct nucleotide can result in sequence read errors in single molecule sequencing by incorporating methods, e.g., because the correct nucleotide can display a longer residence time than an incorrect nucleotide at the site, and can be read by the readout system as an incorporation event. The polymerase kinetic cycle is repeated for the same site, eventually resulting in actual physical incorporation of the correct nucleotide at the site. However, where both the failed incorporation and the actual incorporation of the nucleotides are read by the system as incorporation events, sequences deciphered during single molecule sequencing (SMS) for the incorporation site have an incorrect “insertion” relative to the correct sequence. This phenomenon is termed “branching” because it leads to a “branch” in the sequence (a site where two identical molecules will be read as having different sequences) and can ultimately generate high error rates during single molecule sequencing. In one aspect of the present invention, modification of the polymerase by site-directed mutagenesis is used to lower the frequency of these “branching” events by creating a more tightly structured binding pocket for the (typically non-natural) nucleotides that are incorporated during SMS. Accordingly, as described in this application, mutants were designed to address this issue by modifying various sites in the Φ29 polymerase, predominantly in and around the binding pocket, to create tighter polymerase-analog interactions during an extension reaction. As noted, the “branching fraction” is the proportion of cognate nucleotide (or nucleotide analog, e.g., A488dA4P) dissociation events from the polymerase active site to the total number of events, e.g., the sum of the incorporation events and dissociation events. Desirably, the branching fraction for a polymerase for a given nucleotide or analog of interest (e.g., a labeled nucleotide analog) should be less than 25%, more preferably less than 20%, more preferably less than 15%, yet more preferably less than 10%, or even less than 5%, 1%, or 0.1% of the total interactions, e.g., dissociation events and association events, of the nucleotide analog with the polymerase binding pocket. For example, the branching fraction can be e.g., about 22.5% or less, about 17.5% or less, about 12.5% or less, or about 7.5% or less. In comparison, a wild type Φ29 polymerase exhibits a branching fraction of about >40% for, e.g., a gamma-linked A488dA4 nucleotide analog, wherein ≧40% of the total events with the A488dA4 nucleotide analog in the polymerase binding pocket are dissociation events.

[0121] Alternately or additionally, the polymerases of the invention can display a branching fraction that is 0.5× as high as a wild type parental polymerase (e.g., a wild type Φ29) or less. Preferably, the branching fraction, or optionally rate, is about 0.25× as high as the parental polymerase or less, e.g., about 0.15× as high or less, or even 0.05× as high or less.

[0122] Polymerases with increased stability of a closed polymerase/DNA complex are another feature of the invention. “Increased stability of a closed polymerase-DNA complex” refers to an increased stability of a polymerase when it is bound to a DNA template, e.g., in the presence of a primer or other moiety that can serve as an extension site for the polymerase. This increased stability can be measured as a rate of dissociation of the polymerase from the DNA, e.g., in the presence of a DNA trap (an excess of a competitor molecule that binds to the polymerase once it releases from the DNA, such as an excess of heparin, non-specific DNA, or the like); optionally, the dissociation rate constant (k_off) is determined. As another example, K_d can be measured to assess stability (K_d is an equilibrium constant depending on the binding rate and dissociation rate constants). Improvements of about 30% in complex stability, and preferably about 50%, about 75%, about 100% or more for a polymerase of the invention as compared to a parental (e.g., wild-type) polymerase are desirable. Increases in stability of the complex lead, e.g., to an increase in processivity of the DNA polymerase, which increases the speed and accuracy of sequence reads. Processivity can be defined as the number of bases that can be read without dissociation of the polymerase; here again improvements of about 30% in complex stability, and preferably about 50%, about 75%, about 100% or more are desirable. Improvements in stability can be brought about by selecting mutations that modify amino acid interactions between major domains of the polymerase that wrap around a DNA in the closed conformation (e.g., when the polymerase is wrapped around the DNA template). These domains include the exonuclease domain, the thumb domain, and the TPR2 domain. These include, e.g., residues 68 to 76 and 92 (exonuclease), 405 to 413 (TPR2), and 560-564 (thumb), with the numbering being relative to wild-type Φ29.

[0123] Polymerases that exhibit altered kinetic behavior, particularly a decreased rate for at least one step in the catalytic cycle, form yet another feature of the invention. Such polymerases optionally exhibit comparable rates (e.g., comparable rate constants) for two steps within the catalytic cycle. As is described in greater detail below, by decreasing the rate of certain steps the interaction between the polymerase and nucleotides or nucleotide analogs to be incorporated can be prolonged, increasing detection of incorporation events in single molecule sequencing techniques and improving sequencing accuracy.

[0124] The properties of decreased sequence branching, increased polymerase complex stability (and processivity), and selectively decreased rates are particularly useful in the context of incorporation of labeled nucleotides by the polymerase, e.g., as detected during “sequencing by incorporation” methods (including, e.g., SMS methods). For example, the invention provides, e.g., compositions that include one or more engineered or modified polymerase enzymes optionally with one or more template DNAs and/or labeled or otherwise modified nucleotides or nucleotide analogs, where the composition exhibits decreased branching fraction, increased stability of the closed polymerase-DNA complex and/or improved processivity, and/or decreased rate constant for one or more steps during template dependent polymerase mediated nucleic acid synthesis. Methods, including SMS methods, using these compositions are also provided, as are general methods of making polymerases having the properties noted herein.

[0125] The polymerase mutations and mutational strategies noted herein can be combined with each other and with essentially any other available mutations and mutational strategies to confer additional improvements in, e.g., nucleotide analog specificity, enzyme processivity, improved retention time of labeled nucleotides in polymerase-DNA-nucleotide complexes, and the like. For example, the mutations and mutational strategies herein can be combined with those taught in, e.g., WO 2007/076057 POLYMERASES FOR NUCLEOTIDE ANALOGUE INCORPORATION by Hanzel et al., WO 2008/051530 POLYMERASE ENZYMES AND REAGENTS FOR ENHANCED NUCLEIC ACID SEQUENCING by Rank et al., US 2010/0075332 ENGINEERING POLYMERASES AND REACTION CONDITIONS FOR MODIFIED INCORPORATION PROPERTIES by Patel et al., US 2010/0112645 GENERATION OF MODIFIED POLYMERASES FOR IMPROVED ACCURACY IN SINGLE MOLECULE SEQUENCING by Clark et al., U.S. Ser. No. 61/072,645, filed Mar. 31, 2008, entitled “GENERATION OF POLYMERASES WITH IMPROVED CLOSED COMPLEX STABILITY AND DECREASED BRANCHING RATE” by Sonya Clark et al., U.S. Ser. No. 61/278,041, filed Sep. 30, 2009, entitled “GENERATION OF MODIFIED POLYMERASES FOR IMPROVED ACCURACY IN SINGLE MOLECULE SEQUENCING” by Sonya Clark et al., and U.S. Ser. No. 61/399,108, filed Jul. 6, 2010, entitled “RECOMBINANT POLYMERASES FOR IMPROVED SINGLE MOLECULE SEQUENCING” by Robin Emig et al. This combination of mutations/mutational strategies can be used to impart several simultaneous improvements to a polymerase (e.g., decreased branch fraction formation, improved specificity, improved processivity, altered rates, improved retention time, improved stability of the closed complex, etc.). In addition, polymerases can be further modified for application-specific reasons, such as to increase photostability, e.g., as taught in U.S. patent application Ser. No. 12/384,110 filed Mar. 30, 2009, by Keith Bjornson et al. entitled “Enzymes Resistant to Photodamage,” to improve activity of the enzyme when bound to a surface, as taught, e.g., in WO 2007/075987 ACTIVE SURFACE COUPLED POLYMERASES by Hanzel et al. and WO 2007/076057 PROTEIN ENGINEERING STRATEGIES TO OPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS by Hanzel et al., or to include purification or handling tags as is taught in the cited references and as is common in the art. Similarly, the modified polymerases described herein can be employed in combination with other strategies to improve polymerase performance, for example, reaction conditions for controlling polymerase rate constants such as taught in U.S. patent application Ser. No. 12/414,191 filed Mar. 30, 2009, and entitled “Two slow-step polymerase enzyme systems and methods,” incorporated herein by reference in its entirety for all purposes.

[0126] Specific mutations noted herein can be used alone or in combination with each other and/or with available mutations as described in the references noted above, or can be used in polymerases that lack such previously described mutations. As just one example, essentially any mutation or combination thereof noted herein can be introduced into an L253A/E375Y/A484E/K512Y, E375Y/K512Y/T368F, E375Y/K512Y/T368F/A484E, E375Y/K512Y/A484E Φ29-type polymerase (e.g., Φ29), optionally, an exonuclease-deficient polymerase.

[0127] Accordingly, among other aspects, the present invention provides new polymerases that incorporate nucleotide analogs, such as dye labeled phosphate labeled analogs, into a growing template copy during DNA amplification. These polymerases are modified such that they have decreased branching fraction formation when incorporating the relevant analogs, have improved DNA-polymerase stability or processivity, and/or have altered kinetic properties as compared to corresponding wild-type or other parental polymerases (e.g., polymerases from which modified recombinant polymerases of the invention were derived, e.g., by mutation). The polymerases of the invention can also include any of the additional features for improved specificity, improved processivity, improved retention time, improved surface stability, affinity tagging, and/or the like.

[0128] These new polymerases are particularly well suited to DNA amplification and/or sequencing applications, particularly sequencing protocols that include detection in real time of the incorporation of labeled analogs into DNA amplicons, since the decreased branch fraction, improved complex stability, and/or altered rates can facilitate discrimination of nucleotide incorporation events from non-incorporation events such as transient binding of a mis-matched nucleotide in the active site of the complex, improve processivity, and/or facilitate detection of incorporation events.

Tags and Other Optional Polymerase Features

[0129] The recombinant DNA polymerase optionally includes additional features exogenous or heterologous to the polymerase. For example, the recombinant polymerase optionally includes one or more tags, e.g., purification, substrate binding, or other tags, such as a polyhistidine tag, a HIS-10 tag, a HIS-6 tag, an alanine tag, an Ala10 tag, an Ala 16 tag, a biotinylation tag (e.g., a Btag or variant thereof), a GST tag, a BiTag, an S Tag, a SNAP-tag, an HA tag, a DSB (Sso7D) tag, a lysine tag, a NanoTag, a Cmyc tag, a tag or linker comprising the amino acids glycine and serine, a tag or linker comprising the amino acids glycine, serine, alanine and histidine, a tag or linker comprising the amino acids glycine, arginine, lysine, glutamine and proline, a plurality of polyhistidine tags, a plurality of HIS-10 tags, a plurality of HIS-6 tags, a plurality of alanine tags, a plurality of Ala10 tags, a plurality of Ala16 tags, a plurality of biotinylation tags (e.g., a plurality of Btags or variants thereof), a plurality of GST tags, a plurality of BiTags, a plurality of S Tags, a plurality of SNAP-tags, a plurality of HA tags, a plurality of DSB (Sso7D) tags, a plurality of lysine tags, a plurality of NanoTags, a plurality of Cmyc tags, a plurality of tags or linkers comprising the amino acids glycine and serine, a plurality of tags or linkers comprising the amino acids glycine, serine, alanine and histidine, a plurality of tags or linkers comprising the amino acids glycine, arginine, lysine, glutamine and proline, biotin, avidin, one or more Factor Xa sites, one or more enterokinase sites, thrombin sites, antibodies or antibody domains, antibody fragments, antigens, receptors, receptor domains, receptor fragments, ligands, dyes, acceptors, quenchers, or combinations thereof. The one or more exogenous or heterologous features at the N- and/or C-terminal regions of the polymerase can find use not only for purification purposes, immobilization of the polymerase to a substrate, and the like, but can also be useful for altering one or more kinetic parameters of the polymerase, e.g., slow polymerase translocation.

[0130] The one or more exogenous or heterologous features can be included at the N-terminal region of the polymerase, at the C-terminal region of the polymerase, or both the N-terminal and C-terminal regions of the polymerase. Where the polymerase includes an exogenous or heterologous feature at both the N-terminal and C-terminal regions, the exogenous or heterologous features can be the same (e.g., a polyhistidine tag, e.g., a HIS-10 tag, at both the N- and C-terminal regions) or different (e.g., a Btag at the N-terminal region and a polyhistidine tag, e.g., HIS-10 tag, at the C-terminal region). Optionally, a terminal region (e.g., the N- or C-terminal region) of a polymerase of the invention can comprise two or more exogenous or heterologous features which can be the same or different (e.g., a Btag and a polyhistidine tag at the N-terminal region, a Btag, a polyhistidine tag, and a Factor Xa recognition site at the N-terminal region, and the like). Further details regarding biotinylation tags (including Btag variants, e.g., BtagV7 and the like) can be found in U.S. patent application Ser. No. 12/815,160 entitled BIOTINYLATION TAG PEPTIDES, filed on Jun. 14, 2010, the full disclosure of which is incorporated herein in its entirety for all purposes.

[0131] Table 1 provides example exogenous or heterologous features (e.g., tags, linkers, and the like) that are optionally present in polymerases of the invention. As noted above, polymerases of the invention can include any of these features alone, or in combination with one or more additional features, at the N-terminal and/or C-terminal regions of the polymerase.

[0132]

TABLE 1
Feature Name	Amino Acid Sequence
GST	MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHL
(SEQ ID NO: 84)	YERDEGDKWRNKKFELGLEFPNLPYYIDGDVK
	LTQSMAIIRYIADKHNMLGGCPKERAEISMLE
	GAVLDIRYGVSRIAYSKDFETLKVDFLSKLPE
	MLKMFEDRLCHKTYLNGDHVTHPDFMLYDALD
	VVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKY
	LKSSKYIAWPLQGWQATFGGGDHPPK
Xa	IEGR
(SEQ ID NO: 85)
Btag	GLNDIFEAQKIEWHE
(SEQ ID NO: 86)
BtagV1	GLNDLFHAQKIEWHE
(SEQ ID NO: 87)
BtagV2	GLNDFFNAQKIEWHE
(SEQ ID NO: 88)
BtagV3	GINDLFSAQKIEWHE
(SEQ ID NO: 89)
BtagV4	GINDIFEAQKIEWHE
(SEQ ID NO: 90)
BtagV5	GLNLIFEAQKIEWHE
(SEQ ID NO: 91)
BtagV6	GLNDLFEAQKIEWHE
(SEQ ID NO: 92)
BtagV7	GLNDFFEAQKIEWHE
(SEQ ID NO: 93)
BtagV8	GLNDIVEAQKIEWHE
(SEQ ID NO: 94)
BtagV9	GLNDIFHAQKIEWHE
(SEQ ID NO: 95)
BtagV10	GLNDIFNAQKIEWHE
(SEQ ID NO: 96)
BtagV11	GLNDIFSAQKIEWHE
(SEQ ID NO: 97)
NanoTag	DVEAWLGARVPLVET
(SEQ ID NO: 98)
GSGAAAAAAAAAH	GSGAAAAAAAAAH
(SEQ ID NO: 99)
1942Linker	GGSGGGSGGGSGG
(SEQ ID NO: 100)
Ala10	AAAAAAAAAA
(SEQ ID NO: 101)
GRKKRRQRRRPPQ	GRKKRRQRRRPPQ
(SEQ ID NO: 102)
Ktag(10)	KKKKKKKKKK
(SEQ ID NO: 103)
GSGAAAAAAAAHH	GSGAAAAAAAAHH
(SEQ ID NO: 104)
GSGAAAAAAAHHH	GSGAAAAAAAHHH
(SEQ ID NO: 105)
Cmyc	EQKLISEEDL
(SEQ ID NO: 106)
DSB (Sso7d)	MATVKFKYKGEEKEVDISKIKKVWRVGKMISF
(SEQ ID NO: 107)	TYDEGGGKTGRGAVSEKDAPKELLQMLEKQKK
His6	GHHHHHH
(SEQ ID NO: 108)
His7	GHHHHHHH
(SEQ ID NO: 109)
His8	GHHHHHHHH
(SEQ ID NO: 110)
His9	GHHHHHHHHH
(SEQ ID NO: 111)
His10	HHHHHHHHHH
(SEQ ID NO: 112)
His11	GHHHHHHHHHHH
(SEQ ID NO: 113)
His12	GHHHHHHHHHHHH
(SEQ ID NO: 114)
His13	GHHHHHHHHHHHHH
(SEQ ID NO: 115)
His14	GHHHHHHHHHHHHHH
(SEQ ID NO: 116)
His15	GHHHHHHHHHHHHHHH
(SEQ ID NO: 117)
His16	GHHHHHHHHHHHHHHHH
(SEQ ID NO: 118)
His17	GHHHHHHHHHHHHHHHHH
(SEQ ID NO: 119)
His18	GHHHHHHHHHHHHHHHHHH
(SEQ ID NO: 120)
His19	GHHHHHHHHHHHHHHHHHHH
(SEQ ID NO: 121)
His20	GHHHHHHHHHHHHHHHHHHHH
(SEQ ID NO: 122)
His21	GHHHHHHHHHHHHHHHHHHHHH
(SEQ ID NO: 123)
His22	GHHHHHHHHHHHHHHHHHHHHHH
(SEQ ID NO: 124)
His23	GHHHHHHHHHHHHHHHHHHHHHHH
(SEQ ID NO: 125)
His24	GHHHHHHHHHHHHHHHHHHHHHHHH
(SEQ ID NO: 126)
Ala16	GAAAAAAAAAAAAAAAA
(SEQ ID NO: 127)

[0133] The exogenous or heterologous features can find use, e.g., in the context of binding a polymerase in an active form to a surface, e.g., to orient and/or protect the polymerase active site when the polymerase is bound to a surface. In general, surface binding elements and purification tags that can be added to the polymerase (recombinantly or, e.g., chemically) include, e.g., Btags, polyhistidine tags, HIS-6 tags, biotin, avidin, GST sequences, modified GST sequences, e.g., that are less likely to form dimers, biotin ligase recognition (BiTag) sequences, S tags, SNAP-tags, enterokinase sites, thrombin sites, antibodies or antibody domains, antibody fragments, antigens, receptors, receptor domains, receptor fragments, ligands, dyes, acceptors, quenchers, or combinations thereof.

[0134] The invention includes DNA polymerases that can be coupled to a surface without substantial loss of activity (e.g., in an active form). DNA polymerases can be coupled to the surface through multiple surface coupling domains, which act in concert to increase binding affinity of the polymerase for the surface and to orient the polymerase relative to the surface. For example, the active site can be oriented distal to the surface, thereby making it accessible to a polymerase substrate (template, nucleotides, etc.). This orientation also tends to reduce surface denaturation effects in the region of the active site. In a related aspect, activity of the enzyme can be protected by making the coupling domains large, thereby serving to further insulate the active site from surface binding effects. Further details regarding the immobilization of a polymerase to a surface (e.g., the surface of a zero mode waveguide) in an active form are found in WO 2007/075987 ACTIVE SURFACE COUPLED POLYMERASES by Hanzel et al., and WO 2007/076057 PROTEIN ENGINEERING STRATEGIES TO OPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS by Hanzel et al.

[0135] The polymerases immobilized on a surface in an active form can be coupled to the surface through a plurality of artificial or recombinant surface coupling domains as discussed above, and typically displays a k_cat/K_m (or V_max/K_m) that is at least about 1%, at least about 10%, at least about 25%, at least about 50%, or at least about 75% as high as a corresponding active polymerase in solution.

[0136] Multiple surface binding domains can be added to orient the polypeptide relative to a surface and/or to increase binding of the polymerase to the surface. By binding a surface at two or more sites, through two or more separate tags, the polymerase is held in a relatively fixed orientation with respect to the surface. Additional details on fixing a polymerase to a surface in an active form, attaching tags, and the like are found in WO 2007/075987 ACTIVE SURFACE COUPLED POLYMERASES by Hanzel et al., and WO 2007/076057 PROTEIN ENGINEERING STRATEGIES TO OPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS by Hanzel et al. Further details on attaching tags is available in the art. See, e.g., U.S. Pat. Nos. 5,723,584 and 5,874,239 for additional information on attaching biotinylation peptides to recombinant proteins.

Exemplary Mutation Combinations

[0137] Exemplary polymerase mutation combinations, and optional corresponding exogenous or heterologous features at the N- and/or C-terminal regions of the polymerase are provided in Table 2. Amino acid substitutions and/or insertions are relative to a wild-type Φ29 DNA polymerase (SEQ ID NO:1). Polymerases of the invention (including those provided in Table 2) can include any exogenous or heterologous feature (or combination of such features) at the N- and/or C-terminal region. For example, it will be understood that polymerase mutants in Table 2 that do not include, e.g., a C-terminal polyhistidine tag can be modified to include a polyhistidine tag at the C-terminal region, alone or in combination with any of the exogenous or heterologous features described herein. Certain amino acids are followed by “co”, meaning that the codon of a nucleic acid encoding that amino acid, tag, etc. is optimized for expression in a bacterial cell.

[0138] As will be appreciated, “mutations” with respect to Table 2 and any of the polymerases described herein can comprise one or more amino acid substitutions, deletions, insertions, and the like. Accordingly, certain mutation combinations provided in Table 2 and elsewhere herein include one or more amino acid insertions. For example, “511.1K 511.2S” indicates the insertion of a lysine residue and a serine residue between positions 511 and 512 relative to a wild-type Φ29 DNA polymerase (SEQ ID NO:1), where the lysine immediately follows position 511 and the serine immediately follows the inserted lysine, etc.

[0139]

TABLE 2
SEQ			C-terminal
ID	N-terminal region		region
NO:	feature(s)	Mutations	feature(s)
2	Btag-His10-Xa	N62D E375Y K512Y
3	Btag-His10-Xa	N62D T368F E375Y K512Y
4	Btag.co-His10.co-	N62D T368F E375Y K512Y
	Xa.co
5	Btagco-His10.co-	T368F E375Y K512Y
	Xa.co.
6	Btagco-His10co.	N62D L253A E375Y A484E K512Y
7	Btagco-His10co.	N62D L253A E375Y K512Y
8	Btag-His10.	N62D T368F E375Y A484E K512Y.co	His10
9	Btag-His10-Xa.	N62D T368F E375Y A484E K512Y
10	Btagco.His10co.	N62D L253A E375Y A484E K512Y.co	His10
11	Btagco.His10co.	N62D L253A E375Y A484E K512Y.co	1942Linkco_Ala10co
12	Btagco.His10co	N62D H149M T368F E375Y D510M K512Y D523M.co
13	Btagco.His10co	N62H E375Y A484E E508R K512Y.co	His10
14	Btagco.His10co	D12R N62H T368F E375Y A484E K512Y.co	His10
15	Btagco.His10co.	D12R T368F E375Y A484E E508R 511.1K 511.2S	His10
		512.1G 512.2S.co
16	Btagco.His10co.	D12R T368F E375Y I378W A484E E508R 511.1K	His10
		511.2S 512.1G 512.2S.co
17	Btagco.His10co	Y148A E375Y A484E K512Y.co
18	Btagco.His10co.	N62D A190E E375Y K422A A484E E508R K512Y.co
19	Btagco.His10co.	N62D I93Y T368F T372Y E375Y I378W K478Y	His10
		A484E E508R 511.1K 511.2S K512Y 512.1G
		512.2S.co
20	Btagco-His10co.	N62D T368F E375Y P477Q A484E K512Y
21	Btagco.His10co.	N62D T368F E375Y L384M A484E K512Y.co
22	Btagco-His10.co-	T368F E375Y P477E K512Y
	Xa.co.
23	Btagco-His10co.	A176V T368F E375Y K512Y
24	Btagco.His10co.	T368F E375Y K422R K512Y
25	Btagco.His10co.	N62D E375Y P477Q A484E K512Y.co
26	Btagco-His10.co-	I93F T368F E375Y A484E K512Y
	Xa.co.
27	Btagco.His10co.	L253A E375Y A484E K512Y.co
28	Btagco.His10co.	N62D L253A E375Y E420M A484E K512Y.co
29	Btagco.His10co.	N62D L253A E375Y K422A A484E K512Y.co
30	Btagco.His10co.	N62D L253A E375Y A484E E508K K512Y.co
31	Btagco.His10co.	N62D S215D L253A E375Y A484E K512Y.co
32	Btagco.His10co.	N62D L253T E375Y A484E K512Y.co
33	Btagco.His10co.	N62D L253A Y369H E375Y A484E K512Y.co
34	Btagco.His10co.	N62D L253A Y369G E375Y A484E K512Y.co
35	Btagco.His10co.	N62D L253A Y369L E375Y A484E K512Y.co
36	Btagco.His10co.	N62D L253A E375F A484E K512Y.co
37	Btagco.His10co.	D66R L253A E375Y A484E K512Y.co
38	Btagco.His10co.	N62D L253A E375Y A484E I504R K512Y.co
39	Btagco.His10co.	N62D L253A E375Y A484E D510K K512Y.co
40	Btagco.His10co.	L253A_E375Y_A484E_K512Y.co	His10co
41	BtagV7co.His10co.	L253A_E375Y_A484E_K512Y.co	His10co
42	Btagco.His10co.	L253A E375Y A484E E508R K512Y.co	His10co

[0140] The amino acid sequences of polymerases harboring the exemplary mutation combinations of Table 2 are provided in Tables 3 and 4. Table 3 includes the amino acid sequence of the polymerase portion only. Table 4 includes the polymerase portion of the molecule as well as the one or more exogenous or heterologous feature(s) at the N- and/or C-terminal region of the polymerase.

[0141]

	TABLE 3
	SEQ ID NO	Amino Acid Sequence
	2	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_E375Y_K512Y	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
		HDLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDICEVRYAYRGGFTWLNDRFKE
		KEIGEGMVFDVNSLYPAQMYSRLLPYGEPI
		VFEGKYVWDEDYPLHIQHIRCEFELKEGYI
		PTIQIKRSRFYKGNEYLKSSGGEIADLWLS
		NVDLELMKEHYDLYNVEYISGLKFKATTGL
		FKDFIDKWTYIKTTSYGMKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWAHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	3	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_T368F_E375	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	Y_K512Y.	HDLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTETPDVIKDIVDPLKK
		GYWAHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	4	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_T368F_E371	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	5Y_K52Y.	HDLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWAHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	5	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	T368F_E375Y_K51	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	2Y	HNLKFDGAFLINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDICLGYKGKRKIFITVI
		YDSLKKLPFPVKKIAKDFKLTVLKGDIDYH
		KERPVGYKITPEEYAYIKNDIQIIAEALLI
		QFKQGLDRMTAGSDSLKGFKDIITTKKFKK
		VFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
		KEIGEGMVFDVNSLYPAQMYSRLLPYGEPI
		VFEGKYVWDEDYPLHIQHIRCEFELKEGYI
		PTIQIKRSRFYKGNEYLKSSGGEIADLWLS
		NVDLELMKEHYDLYNVEYISGLKFKATTGL
		FKDFIDKWFYIKTTSYGAIKQLAKLMLNSL
		YGKFASNPDVTGKVPYLKENGALGFRLGEE
		ETKDPVYTPMGVFITAWARYTTITAAQACY
		DRIIYCDTDSIHLTGTEIPDVIKDIVDPKK
		LGYWAHESTFKRAKYLRQKTYIQDIYMKEV
		DGYLVEGSPDDYTDIKFSVKCAGMTDKIKK
		EVTFENFKVGFSRKMKPKPVQVPGGVVLVD
		DTFTIK
	6	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_L253A_E375	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	Y_A484E_K512Y	HDLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMTDICLGYKGKRKIHTVIY
		YDSLKKLPFPVKKIAKDFKLTVLKGDIDYH
		KERPVGYKITPEEYAYIKNDIQIIAEALLI
		QFKQGLDRMTAGSDSLKGFKDIITTKKFKK
		VFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
		KEIGEGMVFDVNSAYPAQMYSRLLPYGEPI
		VFEGKYVWDEDYPLHIQHIRCEFELKEGYI
		PTIQIKRSRFYKGNEYLKSSGGEIADLWLS
		NVDLELMKEHYDLYNVEYISGLKFKATTGL
		FKDFIDKWTYIKTTSYGAIKQLAKLMLNSL
		YGKFASNPDVTGKVPYLKENGALGFRLGEE
		ETKDPVYTPMGVFITAWARYTTITAAQACY
		DRIIYCDTDSIHLTGTEIPDVIKDIVDPKK
		LGYWEHESTFKRAKYLRQKTYIQDIYMKEV
		DGYLVEGSPDDYTDIKFSVKCAGMTDKIKK
		EVTFENFKVGFSRKMKPKPVQVPGGVVLVD
		DTFTIK
	7	MKHNLPRKMYSCDFETTTKVEDCRVWAYGY
	N62D_L253A_E37	MNIEDHSEYKIGNSLDEFMAWVLKVQADLY
	5Y_K512Y	FHDLKFDGAFIINWLERNGFKWSADGLPNT
		YNTHSRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWAHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	8	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_T368F_E375	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	Y_A484E_K512Y.co	HDLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDKLGYKGKRKIHTVIYD
		SLKKLPFPVKKIAKDFKLTVLKGDIDYHKE
		RPVGYKITPEEYAYIKNDIQIIAEALLIQF
		KQGLDRMTAGSDSLKGFKDIITTKKFKKVF
		PTLSLGLDKEVRYAYRGGFTWLNDRFKEKE
		IGEGMVFDVNSLYPAQMYSRLLPYGEPIVF
		EGKYVWDEDYPLHIQHIRCEFELKEGYIPT
		IQIKRSRFYKGNEYLKSSGGEIADLWLSNV
		DLELMKEHYDLYNVEYISGLKFKATTGLFK
		DFIDKWFYIKTTSYGAIKQLAKLMLNSLYG
		KFASNPDVTGKVPYLKENGALGFRLGEEET
		KDPVYTPMGVNTAWARYTTITAAQACYDRI
		IYCDTDSIHLTGTEIPDVIKDIVDPKKLGY
		WEHESTFKRAKYLRQKTYIQDIYMKEVDGY
		LVEGSPDDYTDIKFSVKCAGMTDKIKKEVT
		FENFKVGFSRKMKPKPVQVPGGVVLVDDTF
		TIK
	9	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_T368F_E375	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	Y_A484E_K512Y	HDLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDKLGYKGKRKIHTVIYD
		SLKKLPFPVKKIAKDFICLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTLKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	10	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_L253A_E375	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	Y_A484E_K512Y.	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	11	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_L253A_E375	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	Y_A484E_K512Y.	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	12	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_H149M_T368	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	F_E375Y_D510M_K	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	512Y_D523M.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYMK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWAHESTFKRAKYLRQKTYIQDIYMKEVM
		GYLVEGSPDDYTMIKFSVKCAGMTDKHCKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	13	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62H_E375Y_A484	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	E_E508R_K512Y.	HHLKFDGAFIINWLERNGFKWSADGLPNTY
	co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKRVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	14	MKHMPRKMYSCRFETTTKVEDCRVWAYGYM
	D12R_N62H_T368	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	F_E375Y_A484E_K	HHLKFDGAFIINWLERNGFKWSADGLPNTY
	512Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYEE
		KERPVGYKITPEEYAYIKNDIQHAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIETTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQIHRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	15	MKHMPRKMYSCRFETTTKVEDCRVWAYGYM
	D12R_T368F_E375	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	Y_A484E_E508R_5	HNLKFDGAFIINWLERNGFKWSADGLPNTY
	11.1K_511.2S_51	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
	2.1G_512.2S.co	DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKRVD
		GKSKGSLVEGSPDDYTDIKFSVKCAGMTDK
		IKKEVTFENFKVGFSRKMKPKPVQVPGGVV
		LVDDTFTIK
	16	MKHMPRKMYSCRFETTTKVEDCRVWAYGYM
	D12R_T368F_E375	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	Y_I378W_A484E—	HNLKFDGAFIINWLERNGFKWSADGLPNTY
	E508R511.1K_51	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
	1.2S_512.	DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
	1G_512.2S.co	ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAWKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKRVD
		GKSKGSLVEGSPDDYTDIKFSVKCAGMTDK
		IKKEVTFENFKVGFSRKMKPKPVQVPGGVV
		LVDDTFTIK
	17	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	Y148A_E375Y—	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	A484E_K512Y.co	HNLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDAHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	18	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_A190E_E3	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	75Y_K422A_A48	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	4E_E508R_K-51	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
	2Y.co	DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTEGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TADPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKRVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	19	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_I93Y_T	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	368F_T372Y_E	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	375Y_I378W_	NTYISRMGQWYMIDICLGYKGKRKIHTVIY
	K478Y_A484E—	DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
	E508R_511.1K—	ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
	511.2S_K512	FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
	Y_512.1G_512.	FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
	2S.co	EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKYTSYGAWKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPYKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKRVD
		GKSYGSLVEGSPDDYTDIKFSVKCAGMTDK
		IKKEVTFENFKVGFSRKMKPKPVQVPGGVV
		LVDDTFTIK
	20	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_T368F_E	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	375Y_P477Q_A4	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	84E_K512Y	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAIKQLAKMMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDQKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		VYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		TTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	21	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_T368F_E	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	375Y_L384M_A	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	484E_K512Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAIKQLAKMMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	22	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	T368F_E375Y—	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	P477E_K512Y	HNLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDEKKL
		GYWAHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	23	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	A176V_T368F—	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	E375Y_K512Y	HNLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEVLLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWAHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	24	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	T368F_E375Y_K	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	422R_K512Y	HNLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TRDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWAHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	25	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_E375Y_P4	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	77Q_A484E_K51	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	2Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDQKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	26	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	I93F_T368F_E	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	357Y_A484E_K	HNLKFDGAFIINWLERNGFKWSADGLPNTY
	512Y	NTFISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSLYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		YKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	27	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	L253A_E375Y—	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	A484E_K512Y.co	HNLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	28	MKHMPRKMYSCDFETTTKVEDCRVWAYGYE
	N62D_L253A—	MNIEDHSEYKIGNSLDEFMAWVLKVQADLY
	E375Y_E420M—	YFHDLKFDGAFIINWLERNGFKWSADGLPN
	A484E_K512Y.co	TYNTIISRMGQWYMIDICLGYKGKRKIHTV
		IYDSLKKLPFPVKKIAKDFKLTVLKGDIDY
		HKERPVGYKITPEEYAYIKNDIQIIAEALL
		IQFKQGLDRMTAGSDSLKGFKDIITTKKFK
		KVFPTLSLGLDKEVRYAYRGGFTWLNDRFK
		EKEIGEGMVFDVNSAYPAQMYSRLLPYGEP
		IVFEGKYVWDEDYPLHIQHIRCEFELKEGY
		IPTIQIKRSRFYKGNEYLKSSGGEIADLWL
		SNVDLELMKEHYDLYNVEYISGLKFKATTG
		LFKDFIDKWTYIKTTSYGAIKQLAKLMLNS
		LYGKFASNPDVTGKVPYLKENGALGFRLGE
		EMTKDPVYTPMGVFITAWARYTTITAAQAC
		YDRIIYCDTDSIHLTGTEIPDVIKDIVDPK
		KLGYWEHESTFKRAKYLRQKTYIQDIYMKE
		VDGYLVEGSPDDYTDIKFSVKCAGMTDKIK
		KEVTFENFKVGFSRKMKPKPVQVPGGVVLV
		DDTFTIK
	29	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_L253A_E	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	375Y_K422A_A4	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	84E_K512Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIK
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TADPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	30	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_L253A_E3	ENIEDHSEYKIGNSLDEFMAWVLKVQADLY
	75Y_A484E_E	FHDLKFDGAFIINWLERNGFKWSADGLPNT
	508K_K512Y	YNTIISRMGQWYMIDICLGYKGKRKIHTVI
	.co	YDSLKKLPFPVKKIAKDFKLTVLKGDIDYH
		KERPVGYKITPEEYAYIKNDIQIIAEALLI
		QFKQGLDRMTAGSDSLKGFKDIITTKKFKK
		VFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
		KEIGEGMVFDVNSAYPAQMYSRLLPYGEPI
		VFEGKYVWDEDYPLHIQHIRCEFELKEGYI
		PTIQIKRSRFYKGNEYLKSSGGEIADLWLS
		NVDLELMKEHYDLYNVEYISGLKFKATTGL
		FKDFIDKWTYIKTTSYGAIKQLAKLMLNSL
		YGKFASNPDVTGKVPYLKENGALGFRLGEE
		ETKDPVYTPMGVFITAWARYTTITAAQACY
		DRIIYCDTDSIHLTGTEIPDVIKDIVDPKK
		LGYWEHESTFKRAKYLRQKTYIQDIYMKKV
		DGYLVEGSPDDYTDIKFSVKCAGMTDKIKK
		EVTFENFKVGFSRKMKPKPVQVPGGVVLVD
		DTFTIK
	31	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_S215D_L2	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	53A_E375Y_A48	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	4E_K512Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLDLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	32	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_L253T_E3	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	75Y_A484E_K51	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	2Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSTYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	33	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_L253A_Y3	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	69H_E375Y_A48	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	4E_K512Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTHIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	34	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_L253A_Y3	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	69G_E375Y_A48	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	4E_K512Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTGIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	35	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_L253A_Y3	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	69L_E375Y_A48	IDLKFDGAFIINWLERNGFKWSADGLPNTY
	4E_K512Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTLIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	36	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_L253A_E3	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	75F_A484E_K51	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	12Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSFGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	37	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	D66R_L253A_E3	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	75Y_A484E_K51	HNLKFRGAFIINWLERNGFKWSADGLPNTY
	2Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	38	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_L253A_E3	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	75Y_A484E_I50	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	4R_K512Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDRYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	39	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	N62D_L253A_E3	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	75Y_A484E_D51	HDLKFDGAFIINWLERNGFKWSADGLPNTY
	0K_K512Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVK
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	40	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	L253A_E375Y_A4	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	84E_K512Y.co	HNLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	41	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	L253A_E375Y_A4	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	84E_K512Y.co	HNLKFDGAFIINWLERNGFKWSADGLPNTY
		NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKEVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK
	42	MKHMPRKMYSCDFETTTKVEDCRVWAYGYM
	L253A_E375Y_A4	NIEDHSEYKIGNSLDEFMAWVLKVQADLYF
	84E_E508R_K512	HNLKFDGAFIINWLERNGFKWSADGLPNTY
	Y.co	NTIISRMGQWYMIDICLGYKGKRKIHTVIY
		DSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
		ERPVGYKITPEEYAYIKNDIQIIAEALLIQ
		FKQGLDRMTAGSDSLKGFKDIITTKKFKKV
		FPTLSLGLDKEVRYAYRGGFTWLNDRFKEK
		EIGEGMVFDVNSAYPAQMYSRLLPYGEPIV
		FEGKYVWDEDYPLHIQHIRCEFELKEGYIP
		TIQIKRSRFYKGNEYLKSSGGEIADLWLSN
		VDLELMKEHYDLYNVEYISGLKFKATTGLF
		KDFIDKWTYIKTTSYGAIKQLAKLMLNSLY
		GKFASNPDVTGKVPYLKENGALGFRLGEEE
		TKDPVYTPMGVFITAWARYTTITAAQACYD
		RIIYCDTDSIHLTGTEIPDVIKDIVDPKKL
		GYWEHESTFKRAKYLRQKTYIQDIYMKRVD
		GYLVEGSPDDYTDIKFSVKCAGMTDKIKKE
		VTFENFKVGFSRKMKPKPVQVPGGVVLVDD
		TFTIK

[0142]

TABLE 4
SEQ ID NO	Amino Acid Sequence
43	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btag-His10-	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
Xa.N62D_E375Y_K512Y.	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRF
	YKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYIS
	GLKFKATTGLFKDFIDKWTYIKTTSYGAIKQLAKLMLNSL
	YGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPM
	GVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVI
	KDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVD
	GYLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGF
	SRKMKPKPVQVPGGVVLVDDTFTIK
44	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btag-His10-	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
Xa.N62D_T368F_E375Y_	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
K512Y.	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRF
	YKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYIS
	GLKFKATTGLFKDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
	GKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMG
	VFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIK
	DIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDG
	YLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFS
	RKMKPKPVQVPGGVVLVDDTFTIK
45	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco-His10co-	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
Xaco.N62D_T368F_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
K512Y.	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRF
	YKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYIS
	GLKFKATTGLFKDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
	GKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMG
	VFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIK
	DIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDG
	YLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFS
	RKMKPKPVQVPGGVVLVDDTFTIK
46	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco-His10co-	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
Xaco.T368F_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER
K512Y.	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRF
	YKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYIS
	GLKFKATTGLFKDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
	GKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMG
	VFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIK
	DIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDG
	YLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFS
	RKMKPKPVQVPGGVVLVDDTFTIK
47	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco-His10co-	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
Xaco.N62D_L253A_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
A484E_K512Y.	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
48	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco-His10co-	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
Xaco.N62D_L253A_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
K512Y.	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYW
	AHESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTD
	IKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
49	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btag.His10.Cterm_His10.	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
N62D_T368F_E375Y_A484E—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWFYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIKGHHHHHHHHHH
50	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btag-His10-	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
Xa.N62D_T368F_E375Y_	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
A484E_K512Y.	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWFYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
51	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.Cterm—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
His10.N62D_L253A_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
A484E_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIKGHHHHHHHHHH
52	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.CTerm—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
1942Linkco_Ala10co..N62D—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
L253A_E375Y_A484E—	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
K512Y.co	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIKGGSGGGLGGGLGGAAAAAAAAAA
53	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co..N62D—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
H149M_T368F_E375Y_D510M—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
K512Y_D523M.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYMKERPVGY
	KITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGF
	KDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFK
	EKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWD
	EDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGG
	EIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFK
	DFIDKWFYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTG
	KVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTT
	ITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYW
	AHESTFKRAKYLRQKTYIQDIYMKEVMGYLVEGSPDDYT
	MIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVP
	GGVVLVDDTFTIK
54	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.CTerm—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
His10.N62H_E375Y_A484E—	EYKIGNSLDEFMAWVLKVQADLYFHHLKFDGAFIINWLER
E508R_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVEPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKRVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENEKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIKGHHHHHHHHHH
55	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.CTerm—	RHMKHMPRKMYSCRFETTTKVEDCRVWAYGYMNIEDHS
His10.D12R_N62H_T368F—	EYKIGNSLDEFMAWVLKVQADLYFHHLKFDGAFIINWLER
E375Y_A484E_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKND
	IQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVF
	PTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDV
	NSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCE
	FELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDL
	ELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWFYIKTTS
	YGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALG
	FRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIY
	CDTDSIHLTGTEIPDVIKDIVDPKKLGYWEHESTFKRAKYL
	RQKTYIQDIYMKEVDGYLVEGSPDDYTDIKFSVKCAGMTD
	KIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK
	GHHHHHHHHHH
56	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.CTerm—	RHMKHMPRKMYSCRFETTTKVEDCRVWAYGYMNIEDHS
His10.D12R_T368F_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER
A484E_E508R_511.1K—	NGFKWSADGLPNTYNTIISRMGWQYMIDICLGYKGKRKIH
511.2S_512.1G_512.2S.co	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWFYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKRVDGKSKGSLVEGSPDD
	YTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQ
	VPGGVVLVDDTFTIKGHHHHHHHHHH
57	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.CTerm—	RHMKHMPRKMYSCRFETTTKVEDCRVWAYGYMNIEDHS
His10.D12R_T368F_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER
I378W_A484E_E508R_511.1K—	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
511.2S_512.1G_512.2S.co	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWFYIKTTSYGAWKQLAKLMLNSLYGKFASNPDVTG
	KVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTT
	ITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYW
	EHESTFKRAKYLRQKTYIQDIYMKRVDGKSKGSLVEGSPD
	DYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPV
	QVPGGVVLVDDTFTIKGHHHHHHHHHH
58	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.Y148A—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
E375Y_A484E_K512Y.co	EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER
	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDAHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
59	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
A190E_E375Y_K422A_A484E—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
E508R_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTEGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETADPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKRVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
60	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.CTerm_	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
His10.N62D_193Y_T368F—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
T372Y_E375Y_1378W_K478Y—	NGFKWSADGLPNTYNTYISRMGQWYMIDICLGYKGKRKI
A484E_E508R_511.1K—	HTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGY
511.2S_K512Y_512.1G—	KITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGF
512.2S.co	KDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFK
	EKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWD
	EDYPLHIQHIRCEFELKEGYIPTIQIKCRSRFYKGNEYLKSSGG
	EIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFK
	DFIDKWFYIKYTSYGAWKQLAKLMLNSLYGKFASNPDVT
	GKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYT
	TITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPYKLGY
	WEHESTFKRAKYLRQKTYIQDIYMKRVDGKSYGSLVEGSP
	DDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKP
	VQVPGGVVLVDDTFTIKGHHHHHHHHHH
61	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco-His10co-	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
Xaco.N62D_T368F_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
P477Q_A484E_K512Y	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRF
	YKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYIS
	GLKFKATTGLFKDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
	GKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMG
	VFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIK
	DIVDQKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVDG
	YLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFS
	RKMKPKPVQVPGGVVLVDDTFTIK
62	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.Phi29.	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
N62D_T368F_E375Y_L384M—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
A484E_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWFYIKTTSYGAIKQLAKMMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
63	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco-His10co-	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
Xaco.T368F_E375Y_P477E_	EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER
K512Y.	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWFYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDEKKLGYW
	AHESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTD
	IKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
64	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco-His10co-	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
Xaco.A176V_T368F_E375Y_	EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER
K512Y.	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEVLLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWFYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYW
	AHESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTD
	IKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
65	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco-His10co-	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
Xaco.T368F_E375Y_K422R_	EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER
K512Y.	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWFYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETRDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYW
	AHESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTD
	IKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
66	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
E375Y_P477Q_A484E—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDQKKLGYW
	EHESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTD
	IKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
67	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco-His10co-	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
Xaco.I93F_T368F_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER
A484E_K512Y.	NGFKWSADGLPNTYNTFISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRF
	YKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYIS
	GLKFKATTGLFKDFIDKWFYIKTTSYGAIKQLAKLMLNSLY
	GKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMG
	VFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIK
	DIVDPKKLGYWEHESTFKRAKYLRQKTYIQDIYMKEVDGY
	LVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSR
	KMKPKPVQVPGGVVLVDDTFTIK
68	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.L253A_	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
E375Y_A484E_K512Y.co	EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER
	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
69	MSVDGLNDIFEAQKIEWHEAMGIEHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D_	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
L253A_E375Y_E420M—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
A484E_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
70	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
L253A_E375Y_K422A—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
A484E_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETADPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
71	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
L253A_E375Y_A484E—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
E508K_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKKVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
72	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
S215D_L253A_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
A484E_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLDLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
73	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
L253T_E375Y_A484E—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSTYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
74	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
L253A_Y369H_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
A484E_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTHIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
75	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
L253A_Y369G_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
A484E_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTGIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDIKKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
76	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D—	RHMKHMPRKKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
L253A_Y369L_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
A484E	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTLIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
77	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
L253A_E375F_A484E—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSFGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
78	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.D66R—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
L253A_E375Y_A484E—	EYKIGNSLDEFMAWVLKVQADLYFHNLKFRGAFIINWLER
K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
79	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
L253A_E375Y_A484E—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
I504R_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDRYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
80	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.N62D—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
L253A_E375Y_A484E—	EYKIGNSLDEFMAWVLKVQADLYFHDLKFDGAFIINWLER
D510K_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVKGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIK
81	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.CTerm—	RHMKEIMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
His10.L253A_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER
A484E_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIKGHHHHHHHHHH
82	MSVDGLNDFFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
BtagV7co.His10co.CTerm—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
His10co.L253A_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFINWLER
A484E_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKEVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIKGHHHHHHHHHH
83	MSVDGLNDIFEAQKIEWHEAMGHHHHHHHHHHSSGHIEG
Btagco.His10co.CTerm—	RHMKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHS
His10co.L253A_E375Y—	EYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER
A484E_E508R_K512Y.co	NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIH
	TVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYK
	ITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFK
	DIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKE
	KEIGEGMVFDVNSAYPAQMYSRLLPYGEPIVFEGKYVWDE
	DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGE
	IADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKD
	FIDKWTYIKTTSYGAIKQLAKLMLNSLYGKFASNPDVTGK
	VPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTI
	TAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWE
	HESTFKRAKYLRQKTYIQDIYMKRVDGYLVEGSPDDYTDI
	KFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPG
	GVVLVDDTFTIKGHHHHHHHHHH

[0143] Additional example polymerase mutations relative to a wild-type Φ29 polymerase (SEQ ID NO:1) include: I378W, I93F, I93Y, I364D, E486K, E486R, I378K, P300E, Y315L, P300G, Y315V, D12R, D12M, D66K, D66R, D66M, P129D, T189D, T203D, S215D, S252D, S329D, N330D, F360D, K361D, T427D, T368Y, K361N, W436Y, V514G, P455D, L381E, N387M, 1170F, 1170R, A176E, A176T, A176V, Q180L, F181P, K182P, Q183D, Q183K, L185D, L185K, A190E, A190F, A190L, A190P, A190T, A190V, G191P, L253E, K361P, D365E, D365P, L381F, L381K, L381R, E508R, E508V, D523F, D523L, D523R, E420R, E420M, L384M, K392R, K392M, K392W, K422M, K422W, F137N, T204E, E508R, 511.1G_—511.2S, 512.1G_—512.2S, 511.1K_—511.2S, K512.1G_—512.2K, 507.1E_—507.2V_—507.3D_—507.4G_—507.5Y, and 511.1E_—511.2V_—511.3D_—511.4G. These mutations, as well as the mutation combinations of Tables 2-4, can be combined with any of the mutations described hereinbelow, e.g., mutations that increase the closed complex stability of a polymerase, mutations that decrease the branching fraction of a polymerase, and/or mutations that reduce reaction rates at one or more steps of the polymerase kinetic cycle.

DNA Polymerases

[0144] A variety of polymerases are generally available and can be modified in a manner similar to those, e.g., in Tables 2-4,6,9 and 10 to confer desirable features (e.g., decreased branching fraction, increased stability for the closed DNA polymerase-DNA complex, altered rate constants) for applications such as nucleic acid amplification and sequencing. DNA polymerases are sometimes classified into six main groups based upon various phylogenetic relationships, e.g., with E. coli Pol I (class A), E. coli Pol II (class B), E. coli Pol III (class C), Euryarchaeotic Pol II (class D), human Pol beta (class X), and E. coli UmuC/DinB and eukaryotic RAD30/xeroderma pigmentosum variant (class Y). For a review of recent nomenclature, see, e.g., Burgers et al. (2001) “Eukaryotic DNA polymerases: proposal for a revised nomenclature” J Biol Chem. 276(47):43487-90. For a review of polymerases, see, e.g., Hübscher et al. (2002) “Eukaryotic DNA Polymerases” Annual Review of Biochemistry Vol. 71: 133-163; Alba (2001) “Protein Family Review: Replicative DNA Polymerases” Genome Biology 2(1):reviews 3002.1-3002.4; and Steitz (1999) “DNA polymerases: structural diversity and common mechanisms” J Biol Chem 274:17395-17398. The basic mechanisms of action for many polymerases have been determined. The sequences of literally hundreds of polymerases are publicly available, and the crystal structures for many of these have been determined, or can be inferred based upon similarity to solved crystal structures for homologous polymerases. For example, the crystal structure of Φ29, a preferred type of parental enzyme to be modified according to the invention, is available.

[0145] In addition to wild-type polymerases, chimeric polymerases made from a mosaic of different sources can be used. For example, chimeric polymerases, where the polymerase is a chimera between a modified Φ29 polymerase (e.g., a Φ29 polymerase that includes a mutation or combination of mutations set forth in Tables 2, 3, and 4) and a polymerase other than a Φ29 polymerase are provided by the present invention. Φ29 polymerases made by taking sequences from more than one parental polymerase into account can be used as a starting point for mutation to produce the polymerases of the invention. Chimeras can be produced, e.g., using consideration of similarity regions between the polymerases to define consensus sequences that are used in the chimera, or using gene shuffling technologies in which multiple Φ29-related polymerases are randomly or semi-randomly shuffled via available gene shuffling techniques (e.g., via “family gene shuffling”; see Crameri et al. (1998) “DNA shuffling of a family of genes from diverse species accelerates directed evolution” Nature 391:288-291; Clackson et al. (1991) “Making antibody fragments using phage display libraries” Nature 352:624-628; Gibbs et al. (2001) “Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling” Gene 271:13-20; and Hiraga and Arnold (2003) “General method for sequence-independent site-directed chimeragenesis: J. Mol. Biol. 330:287-296). In these methods, the recombination points can be predetermined such that the gene fragments assemble in the correct order. However, the combinations, e.g., chimeras, can be formed at random. For example, using methods described in Clarkson et al., five gene chimeras, e.g., comprising segments of a Phi29 polymerase, a PZA polymerase, a M2 polymerase, a B103 polymerase, and a GA-1 polymerase, can be generated. Appropriate mutations to improve branching fraction, increase closed complex stability, or alter reaction rate constants can be introduced into the chimeras.

[0146] Available DNA polymerase enzymes have also been modified in any of a variety of ways, e.g., to reduce or eliminate exonuclease activities (many native DNA polymerases have a proof-reading exonuclease function that interferes with, e.g., sequencing applications), to simplify production by making protease digested enzyme fragments such as the Klenow fragment recombinant, etc. As noted, polymerases have also been modified to confer improvements in specificity, processivity, and improved retention time of labeled nucleotides in polymerase-DNA-nucleotide complexes (e.g., WO 2007/076057 POLYMERASES FOR NUCLEOTIDE ANALOGUE INCORPORATION by Hanzel et al., WO 2008/051530 POLYMERASE ENZYMES AND REAGENTS FOR ENHANCED NUCLEIC ACID SEQUENCING by Rank et al., US 2010/0112645 GENERATION OF MODIFIED POLYMERASES FOR IMPROVED ACCURACY IN SINGLE MOLECULE SEQUENCING by Clark et al.), to alter branch fraction and translocation (e.g., U.S. patent application Ser. No. 12/584,481 filed Sep. 4, 2009, by Pranav Patel et al. entitled “ENGINEERING POLYMERASES AND REACTION CONDITIONS FOR MODIFIED INCORPORATION PROPERTIES”), to increase photostability (e.g., U.S. patent application Ser. No. 12/384,110 filed Mar. 30, 2009, by Keith Bjornson et al. entitled “Enzymes Resistant to Photodamage”), and to improve surface-immobilized enzyme activities (e.g., WO 2007/075987 ACTIVE SURFACE COUPLED POLYMERASES by Hanzel et al. and WO 2007/076057 PROTEIN ENGINEERING STRATEGIES TO OPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS by Hanzel et al.). Any of these available polymerases can be modified in accordance with the invention to decrease branching fraction formation, improve stability of the closed polymerase-DNA complex, and/or alter reaction rate constants.

[0147] Many such polymerases that are suitable for modification are available, e.g., for use in sequencing, labeling and amplification technologies. For example, human DNA Polymerase Beta is available from R&D systems. DNA polymerase I is available from Epicenter, GE Health Care, Invitrogen, New England Biolabs, Promega, Roche Applied Science, Sigma Aldrich and many others. The Klenow fragment of DNA Polymerase I is available in both recombinant and protease digested versions, from, e.g., Ambion, Chimerx, eEnzyme LLC, GE Health Care, Invitrogen, New England Biolabs, Promega, Roche Applied Science, Sigma Aldrich and many others. Φ29 DNA polymerase is available from e.g., Epicentre. Poly A polymerase, reverse transcriptase, Sequenase, SP6 DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, and a variety of thermostable DNA polymerases (Taq, hot start, titanium Taq, etc.) are available from a variety of these and other sources. Recent commercial DNA polymerases include Phusion™ High-Fidelity DNA Polymerase, available from New England Biolabs; GoTaq® Flexi DNA Polymerase, available from Promega; RepliPHI™ Φ29 DNA Polymerase, available from Epicentre Biotechnologies; PfuUltra™ Hotstart DNA Polymerase, available from Stratagene; KOD HiFi DNA Polymerase, available from Novagen; and many others. Biocompare(dot)com provides comparisons of many different commercially available polymerases.

[0148] DNA polymerases that are preferred substrates for mutation to decrease branching fraction, increase closed complex stability, or alter reaction rate constants include Taq polymerases, exonuclease deficient Taq polymerases, E. coli DNA Polymerase 1, Klenow fragment, reverse transcriptases, Φ29 related polymerases including wild type Φ29 polymerase and derivatives of such polymerases such as exonuclease deficient forms, T7 DNA polymerase, T5 DNA polymerase, an RB69 polymerase, etc.

[0149] In one aspect, the polymerase that is modified is a Φ29-type DNA polymerase. For example, the modified recombinant DNA polymerase can be homologous to a wild-type or exonuclease deficient Φ29 DNA polymerase, e.g., as described in U.S. Pat. Nos. 5,001,050, 5,198,543, or 5,576,204. Alternately, the modified recombinant DNA polymerase can be homologous to other Φ29-type DNA polymerases, such as B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, Φ21, or the like. For nomenclature, see also, Meijer et al. (2001) “Φ29 Family of Phages” Microbiology and Molecular Biology Reviews, 65(2):261-287.

Nucleotide Analogs

[0150] As discussed, various polymerases of the invention can incorporate one or more nucleotide analogs into a growing oligonucleotide chain. Upon incorporation, the analog can leave a residue that is the same as or different than a natural nucleotide in the growing oligonucleotide (the polymerase can incorporate any non-standard moiety of the analog, or can cleave it off during incorporation into the oligonucleotide). A “nucleotide analog” (or “nucleotide analogue”) herein is a compound, that, in a particular application, functions in a manner similar or analogous to a naturally occurring nucleoside triphosphate (a “nucleotide”), and does not otherwise denote any particular structure. A nucleotide analog is an analog other than a standard naturally occurring nucleotide, i.e., other than A, G, C, T, or U, though upon incorporation into the oligonucleotide, the resulting residue in the oligonucleotide can be the same as (or different from) an A, G, C, T, or U residue.

[0151] In one useful aspect of the invention, nucleotide analogs can also be modified to achieve any of the improved properties desired. For example, various linkers or other substituents can be incorporated into analogs that have the effect of reducing branching fraction, improving processivity, or altering rates. Modifications to the analogs can include extending the phosphate chains, e.g., to include a tetra-, penta-, hexa- or heptaphosphate group, and/or adding chemical linkers to extend the distance between the nucleotide base and the dye molecule, e.g., a fluorescent dye molecule. Substitution of one or more non-bridging oxygen in the polyphosphate, for example with S or BH₃, can change the polymerase reaction kinetics, e.g., to achieve a system having two slow steps as described hereinbelow. Optionally, one or more, two or more, three or more, or four or more non-bridging oxygen atoms in the polyphosphate group of the analog has an S substituted for an O. While not being bound by theory, it is believed that the properties of the nucleotide, such as the metal chelation properties, electronegativity, or steric properties, can be altered by substitution of the non-bridging oxygen(s).

[0152] Many nucleotide analogs are available and can be incorporated by the polymerases of the invention. These include analog structures with core similarity to naturally occurring nucleotides, such as those that comprise one or more substituent on a phosphate, sugar or base moiety of the nucleoside or nucleotide relative to a naturally occurring nucleoside or nucleotide. In one embodiment, the nucleotide analog includes three phosphate containing groups; for example, the analog can be a labeled nucleoside triphosphate analog and/or an α-thiophosphate nucleotide analog having three phosphate groups. In one embodiment, a nucleotide analog can include one or more extra phosphate containing groups, relative to a nucleoside triphosphate. For example, a variety of nucleotide analogs that comprise, e.g., from 4-6 or more phosphates are described in detail in U.S. patent application Ser. No. 11/241,809, filed Sep. 29, 2005, and incorporated herein by reference in its entirety for all purposes. Other exemplary useful analogs, including tetraphosphate and pentaphosphate analogs, are described in U.S. Pat. No. 7,041,812, incorporated herein by reference in its entirety for all purposes.

[0153] For example, the analog can include a labeled compound of the formula:

[0154]
wherein B is a nucleobase (and optionally includes a label); S is selected from a sugar moiety, an acyclic moiety or a carbocyclic moiety (and optionally includes a label); L is an optional detectable label; R₁ is selected from O and S; R₂, R₃ and R₄ are independently selected from O, NH, S, methylene, substituted methylene, C(O), C(CH₂), CNH₂, CH₂CH₂, C(OH)CH₂R where R is 4-pyridine or 1-imidazole, provided that R₄ may additionally be selected from

[0155]
and

[0156]
R₅, R₆, R₇, R₈, R₁₁ and R₁₃ are, when present, each independently selected from O, BH₃, and S; and R₉, R₁₀ and R₁₂ are independently selected from O, NH, S, methylene, substituted methylene, CNH₂, CH₂CH₂, C(OH)CH₂R where R is 4-pyridine or 1-imidazole. In some cases, phosphonate analogs may be employed as the analogs, e.g., where one of R₂, R₃, R₄, R₉, R₁₀ or R₁₂ are not O, e.g., they are methyl etc. See, e.g., U.S. patent application Ser. No. 11/241,809, previously incorporated herein by reference in its entirety for all purposes.

[0157] The base moiety incorporated into the analog is generally selected from any of the natural or non-natural nucleobases or nucleobase analogs, including, e.g., purine or pyrimidine bases that are routinely found in nucleic acids and available nucleic acid analogs, including adenine, thymine, guanine, cytidine, uracil, and in some cases, inosine. As noted, the base optionally includes a label moiety. For convenience, nucleotides and nucleotide analogs are generally referred to based upon their relative analogy to naturally occurring nucleotides. As such, an analog that operates, functionally, like adenosine triphosphate, may be generally referred to herein by the shorthand letter A. Likewise, the standard abbreviations of T, G, C, U and I, may be used in referring to analogs of naturally occurring nucleosides and nucleotides typically abbreviated in the same fashion. In some cases, a base may function in a more universal fashion, e.g., functioning like any of the purine bases in being able to hybridize with any pyrimidine base, or vice versa. The base moieties used in the present invention may include the conventional bases described herein or they may include such bases substituted at one or more side groups, or other fluorescent bases or base analogs, such as 1,N6 ethenoadenosine or pyrrolo C, in which an additional ring structure renders the B group neither a purine nor a pyrimidine. For example, in certain cases, it may be desirable to substitute one or more side groups of the base moiety with a labeling group or a component of a labeling group, such as one of a donor or acceptor fluorophore, or other labeling group. Examples of labeled nucleobases and processes for labeling such groups are described in, e.g., U.S. Pat. Nos. 5,328,824 and 5,476,928, each of which is incorporated herein by reference in its entirety for all purposes.

[0158] In the analogs, the S group is optionally a sugar moiety that provides a suitable backbone for a synthesizing nucleic acid strand. For example, the sugar moiety is optionally selected from a D-ribosyl, 2′ or 3′ D-deoxyribosyl, 2′,3′-D-dideoxyribosyl, 2′, 3′-D-didehydrodideoxyribosyl, 2′ or 3′ alkoxyribosyl, 2′ or 3′ aminoribosyl, 2′ or 3′ mercaptoribosyl, 2′ or 3′ alkothioribosyl, acyclic, carbocyclic or other modified sugar moieties. A variety of carbocyclic or acyclic moieties can be incorporated as the “S” group in place of a sugar moiety, including, e.g., those described in U.S. Patent Application Publication No. 2003/0124576, which is incorporated herein by reference in its entirety for all purposes.

[0159] For most cases, the phosphorus containing chain in the analogs, e.g., a triphosphate in conventional NTPs, is preferably coupled to the 5′ hydroxyl group, as in natural nucleoside triphosphates. However, in some cases, the phosphorus containing chain is linked to the S group by the 3′ hydroxyl group.

[0160] L generally refers to a detectable labeling group that is coupled to the terminal phosphorus atom via the R₄ (or R₁₀ or R₁₂ etc.) group. The labeling groups employed in the analogs of the invention may comprise any of a variety of detectable labels. Detectable labels generally denote a chemical moiety that provides a basis for detection of the analog compound separate and apart from the same compound lacking such a labeling group. Examples of labels include, e.g., optical labels, e.g., labels that impart a detectable optical property to the analog, electrochemical labels, e.g., labels that impart a detectable electrical or electrochemical property to the analog, and physical labels, e.g., labels that impart a different physical or spatial property to the analog, e.g., a mass tag or molecular volume tag. In some cases individual labels or combinations may be used that impart more than one of the aforementioned properties to the analogs of the invention.

[0161] Optionally, the labeling groups incorporated into the analogs comprise optically detectable moieties, such as luminescent, chemiluminescent, fluorescent, fluorogenic, chromophoric and/or chromogenic moieties, with fluorescent and/or fluorogenic labels being preferred. A variety of different label moieties are readily employed in nucleotide analogs. Such groups include fluorescein labels, rhodamine labels, cyanine labels (i.e., Cy3, Cy5, and the like, generally available from the Amersham Biosciences division of GE Healthcare), the Alexa family of fluorescent dyes and other fluorescent and fluorogenic dyes available from Molecular Probes/Invitrogen, Inc. and described in ‘The Handbook—A Guide to Fluorescent Probes and Labeling Technologies, Tenth Edition’ (2005) (available from Invitrogen, Inc./Molecular Probes). A variety of other fluorescent and fluorogenic labels for use with nucleoside polyphosphates, and which would be applicable to the nucleotide analogs incorporated by the polymerases of the present invention, are described in, e.g., U.S. Patent Application Publication No. 2003/0124576, previously incorporated herein by reference in its entirety for all purposes.

[0162] Additional details regarding analogs and methods of making such analogs can be found in U.S. patent application Ser. No. 11/241,809, filed Sep. 29, 2005, and incorporated herein by reference in its entirety for all purposes.

[0163] Thus, in one illustrative example, the analog can be a phosphate analog (e.g., an analog that has more than the typical number of phosphates found in nucleoside triphosphates) that includes, e.g., an Alexa dye label. For example, an Alexa488 dye can be labeled on a delta phosphate of a tetraphosphate analog (denoted, e.g., A488dC4P or A488dA4P, shown in FIG. 3, for the Alexa488 labeled tetraphosphate analogs of C and A, respectively), or an Alexa568 or Alexa633 dye can be used (e.g., A568dC4P and A633dC4P, respectively, for labeled tetraphosphate analogs of C or A568dT6P for a labeled hexaphospate analog of T), or an Alexa546 dye can be used (e.g., A546dG4P), or an Alexa594 dye can be used (e.g., A594dT4P). As additional examples, an Alexa555 dye (e.g., A555dC6P or A555dA6P), an Alexa 647 dye (e.g., A647dG6P), an Alexa 568 dye (e.g., A568dT6P), and/or an Alexa660 dye (e.g., A660dA6P or A660dC6P) can be used in, e.g., single molecule sequencing. Similarly, to facilitate color separation, a pair of fluorophores exhibiting FRET (fluorescence resonance energy transfer) can be labeled on a delta phosphate of a tetraphosphate analog (denoted, e.g., FAM-amb-A532dG4P or FAM-amb-A594dT4P).

Applications for Enhanced Nucleic Acid Amplification and Sequencing

[0164] Polymerases of the invention, e.g., modified recombinant polymerases, are optionally used in combination with nucleotides and/or nucleotide analogs and nucleic acid templates (DNA or RNA) to copy the template nucleic acid. That is, a mixture of the polymerase, nucleotides/analogs, and optionally other appropriate reagents, the template and a replication initiating moiety (e.g., primer) is reacted such that the polymerase synthesizes nucleic acid (e.g., extends the primer) in a template-dependent manner. The replication initiating moiety can be a standard oligonucleotide primer, or, alternatively, a component of the template, e.g., the template can be a self-priming single stranded DNA, a nicked double stranded DNA, or the like. Similarly, a terminal protein can serve as a initiating moiety. At least one nucleotide analog can be incorporated into the DNA. The template DNA can be a linear or circular DNA, and in certain applications, is desirably a circular template (e.g., for rolling circle replication or for sequencing of circular templates). Optionally, the composition can be present in an automated DNA replication and/or sequencing system.

[0165] Incorporation of labeled nucleotide analogs by the polymerases of the invention is particularly useful in a variety of different nucleic acid analyses, including real-time monitoring of DNA polymerization. The label can itself be incorporated, or more preferably, can be released during incorporation of the analog. For example, analog incorporation can be monitored in real-time by monitoring label release during incorporation of the analog by the polymerase. The portion of the analog that is incorporated can be the same as a natural nucleotide, or can include features of the analog that differ from a natural nucleotide.

[0166] In general, label incorporation or release can be used to indicate the presence and composition of a growing nucleic acid strand, e.g., providing evidence of template replication/amplification and/or sequence of the template. Signaling from the incorporation can be the result of detecting labeling groups that are liberated from the incorporated analog, e.g., in a solid phase assay, or can arise upon the incorporation reaction. For example, in the case of FRET labels where a bound label is quenched and a free label is not, release of a label group from the incorporated analog can give rise to a fluorescent signal. Alternatively, the enzyme may be labeled with one member of a FRET pair proximal to the active site, and incorporation of an analog bearing the other member will allow energy transfer upon incorporation. The use of enzyme bound FRET components in nucleic acid sequencing applications is described, e.g., in U.S. Patent Application Publication No. 2003/0044781, incorporated herein by reference.

[0167] In one example reaction of interest, a polymerase reaction can be isolated within an extremely small observation volume that effectively results in observation of individual polymerase molecules. As a result, the incorporation event provides observation of an incorporating nucleotide analog that is readily distinguishable from non-incorporated nucleotide analogs. In a preferred aspect, such small observation volumes are provided by immobilizing the polymerase enzyme within an optical confinement, such as a Zero Mode Waveguide (ZMW). For a description of ZMWs and their application in single molecule analyses, and particularly nucleic acid sequencing, see, e.g., U.S. Patent Application Publication No. 2003/0044781, and U.S. Pat. No. 6,917,726, each of which is incorporated herein by reference in its entirety for all purposes. See also Levene et al. (2003) “Zero-mode waveguides for single-molecule analysis at high concentrations” Science 299:682-686, Eid et al. (2009) “Real-time DNA sequencing from single polymerase molecules” Science 323:133-138, and U.S. Pat. Nos. 7,056,676, 7,056,661, 7,052,847, and 7,033,764, the full disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0168] In general, a polymerase enzyme is complexed with the template strand in the presence of one or more nucleotides and/or one or more nucleotide analogs. For example, in certain embodiments, labeled analogs are present representing analogous compounds to each of the four natural nucleotides, A, T, G and C, e.g., in separate polymerase reactions, as in classical Sanger sequencing, or multiplexed together, e.g., in a single reaction, as in multiplexed sequencing approaches. When a particular base in the template strand is encountered by the polymerase during the polymerization reaction, it complexes with an available analog that is complementary to such nucleotide, and incorporates that analog into the nascent and growing nucleic acid strand. In one aspect, incorporation can result in a label being released, e.g., in polyphosphate analogs, cleaving between the α and β phosphorus atoms in the analog, and consequently releasing the labeling group (or a portion thereof). The incorporation event is detected, either by virtue of a longer presence of the analog and, thus, the label, in the complex, or by virtue of release of the label group into the surrounding medium. Where different labeling groups are used for each of the types of analogs, e.g., A, T, G or C, identification of a label of an incorporated analog allows identification of that analog and consequently, determination of the complementary nucleotide in the template strand being processed at that time. Sequential reaction and monitoring permits real-time monitoring of the polymerization reaction and determination of the sequence of the template nucleic acid. As noted above, in particularly preferred aspects, the polymerase enzyme/template complex is provided immobilized within an optical confinement that permits observation of an individual complex, e.g., a zero mode waveguide. For additional information on single molecule sequencing monitoring incorporation of phosphate-labeled analogs in real time, see, e.g., Eid et al. (2009) “Real-time DNA sequencing from single polymerase molecules” Science 323:133-138.

[0169] In addition to their use in sequencing, the polymerases of the invention are also useful in a variety of other genotyping analyses, e.g., SNP genotyping using single base extension methods, real time monitoring of amplification, e.g., RT-PCR methods, and the like. Further details regarding sequencing and nucleic acid amplification can be found, e.g., in Sambrook, Ausubel, and Innis, all infra.

Modified Recombinant Polymerases with Increased Closed Complex Stability

[0170] The invention features methods of generating recombinant DNA polymerases with modifications that increase the stability of the closed polymerase/DNA complex, compositions that include such polymerases, and methods of using such modified polymerases to, e.g., sequence a DNA template or make a DNA. Any of a number of polymerases, e.g., those described herein or polymerases homologous to those described herein, can be modified to exhibit increased closed polymerase/DNA complex stability using the methods described herein. In a preferred embodiment, a Φ29 polymerase and Φ29 polymerase derivatives, e.g., exonuclease-deficient Φ29 mutants, Φ29-type polymerases, or polymerases homologous to Φ29, can be modified to exhibit this phenotype. For example, any of the mutations described herein for increasing the closed complex stability of a polymerase can be present in, or added to, e.g., any of the polymerases of Tables 2-4 to modulate the closed complex stability of such polymerases. Moreover, the mutations provided in Tables 2-4, alone or in combination, can be added to polymerases that include mutations for increased closed complex stability described herein.

[0171] A closed polymerase/DNA complex is formed, e.g., by Φ29 DNA polymerase, when the Terminal Protein Region 2 (TPR2), exonuclease, thumb, and palm subdomains of Φ29 (FIG. 1 Panels A and B) encircle the DNA binding groove at the polymerization active site, forming a “doughnut” (FIG. 1 Panel B and FIG. 2 Panel A) around the upstream duplex DNA. This conformation enhances polymerase processivity in a manner analogous to sliding clamp proteins (Kamtekar, et al. (2004) “Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29.” Mol. Cell 16: 1035-6). The other Φ29 subdomains represented in FIG. 1 include TPR1 and fingers. It is worth noting that closed complex formation can be independent of the presence of a nucleotide or nucleotide analog.

[0172] Φ29 DNA polymerase mutants lacking the TPR2 subdomain exhibit drastically decreased processivity (Rodriguez, et al. (2005) “A specific subdomain in Φ29 polymerase confers both processivity and strand-displacement capacity” Proc Natl Acad Sci USA 102: 6407-6412), indicating that mutations that stabilize the protein-protein interactions at the interface of these subdomains (FIG. 2 Panel B, example circled) can increase the stability of the closed complex comprising the polymerase and DNA, e.g., a template strand and a primer. An increase in closed polymerase/DNA complex stability can comprise an improvement of at least 30%, e.g., 50% or better, 75% or better, or even 100% or better.

[0173] Mutations that increase the stability of the closed polymerase/DNA complex can indirectly improve polymerase processivity and can generate polymerases that can be of beneficial use in any application where increased read length, speed and accuracy of polymerization is desirable, e.g., single-molecule sequencing (SMS), e.g., in a zero-mode waveguide (ZMW), SNP genotyping using single base extension methods, real time monitoring of amplification, e.g., RT-PCR methods, and the like. Useful compositions comprising such polymerases can include nucleotide analogs, e.g., analogs labeled with fluorophores, phosphate-labeled nucleotide analogs, and/or labeled nucleotide analogs having, e.g., 3-7 phosphate groups, that the polymerase can incorporate into a DNA. In some embodiments of the compositions, a modified polymerase with improved closed polymerase/DNA complex stability can be immobilized on a surface, e.g., in a ZMW.

[0174] Mutations that can stabilize a closed polymerase/DNA complex include mutations to amino acids regions that correspond to Ala68-Arg76, Tyr405-Gly413, and Gln560-Gly564 of wild type Φ29. These amino acid regions comprise the interface of the exonuclease, TPR2, and thumb subdomains, respectively, and are depicted in FIG. 2 Panels A and B. Mutation of Thr92, in the exonuclease domain, can also stabilize interaction with TPR2 domain. Mutations can be introduced into one or more of these residues to provide additional stability to the closed complex, e.g., by stabilizing the interface of the exonuclease, TPR2, and thumb domains. For example, the hydrophobic environment between domains can be increased to increase complex stability, charged residues can be introduced to add favorable electrostatic interactions (or removed to remove unfavorable interactions), hydrogen bonds can be introduced, and the like. In general terms, a mutation can introduce an intramolecular interaction between domains that is predicted to stabilize the interface (and thus the closed complex) and/or can remove an interaction predicted to destabilize the interface. Thus, strategic mutations such as Thr92Phe, Thr92Ile, Gly410Asp, Asn72Ala, Asn72Ile, Asn72Phe, or Asn72Ser, or combinations thereof such as Thr92Ile and Gly104Asp, can stabilize a closed polymerase/DNA complex. Such mutations can be present in, or added to, e.g., the polymerases of Tables 2, 3 and 4 to stabilize a closed polymerase/DNA complex. The modified polymerases can include, e.g., L253A, T368F, E375Y, A484E, and/or K512Y substitutions or other mutations described herein. Moreover, the mutations provided in Tables 2-4, alone or in combination, can be added to polymerases that include mutations for increased closed complex stability described herein. Strategies for mutating and screening polymerases are detailed herein.

[0175] Increases in the stability of a closed polymerase/DNA complex can be measured by comparing a rate of dissociation or the dissociation rate constant (k_off) of the modified polymerase from a DNA to k_off of the parental polymerase from a DNA. Decreases in k_off can correspond to an increase in closed complex stability. In one preferred embodiment, k_off can be determined by, e.g., stopped-flow fluorometric analysis, incubating a fluorescently labeled DNA template, e.g., 2-aminopurine-labeled DNA, with a modified polymerase in the presence of an excess of competitor, e.g., unlabelled DNA or heparin. In another embodiment, a preformed complex comprising a modified polymerase and a template DNA can be incubated in the presence of excess competitor DNA or heparin. A time course of activity assays, e.g., primer extension, can measure the fraction of polymerase that remains associated with template. As indicated above, k_off is optionally decreased by at least 30%, e.g., by at least 50%, at least 75%, or at least 100%, for the modified recombinant polymerase as compared to the parental polymerase.

[0176] Increases in the stability of a closed polymerase/DNA complex can also be measured by determining the equilibrium dissociation constant K_d, where a decrease in K_d can correspond to increased closed complex stability. Optionally, K_d is decreased by at least 30%, e.g., by at least 50%, at least 75%, or at least 100%, for the modified recombinant polymerase as compared to the parental polymerase. K_d can be determined using techniques known in the art, for example, surface plasmon resonance (SPR), fluorescent anisotropy measurements, gel mobility shift assays, or isothermal titration calorimetry (ITC).

[0177] Processivity can be defined as the modified polymerase's extension rate constant (k_ext) divided by the sum of the extension rate constant and the rate constant for dissociation of the modified polymerase from a DNA (k_off), e.g., k_ext/(k_ext+k_off). As described herein, mutations in a polymerase that improve the stability of a closed polymerase/DNA complex can result in a measurable decrease in k_off, which can, accordingly, improve the polymerase's processivity, such that the modified polymerase's processivity is, e.g., at least twice that of the polymerase from which is was derived, or better. In a related aspect, a modified polymerase's processivity can be improved by increasing its extension rate, a phenotype which can be dependent on the type of nucleotide and/or nucleotide analog assayed. The extension rate constant can be determined using techniques known in the art. See, e.g., Korlach et al. (2008) “Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides” Nucleosides Nucleotides Nucleic Acids 27(9):1072-83 (defined as k_el).

[0178] It will be understood that any of the above mutations that increase the closed complex stability of a polymerase can be combined with, e.g., the mutation or mutation combinations provided in Tables 2-4, the mutations described herein that decrease the branching fraction of a polymerase, and/or the mutations described herein that reduce reaction rates at one or more steps of the polymerase kinetic cycle.

Modified Recombinant Polymerases with Decreased Branching Fraction

[0179] During a polymerase kinetic cycle, sampling of each of the possible nucleotides or nucleotide analogs occurs until a correct Watson-Crick pairing is generated (see, e.g., Hanzel, et al. WO 2007/076057 POLYMERASES FOR NUCLEOTIDE ANALOGUE INCORPORATION for a description of the kinetic cycle of a polymerase; see also the section entitled “Polymerase Mediated Synthesis” hereinbelow). According to structural studies of DNA polymerases complexed with DNA substrates, the primer-terminus can not typically form a covalent bond with an incorrectly paired nucleotide (Berman, et al. (2007) “Structures of phi29 polymerase complexed with substrate: the mechanism of translocation in polymerases.” EMBO J 26: 3494-3505). Chemical linkages between a correctly paired nucleotide and the 3′OH of a preceding base can also fail to form, e.g., due to premature release of the sampled nucleotide from the active site. Sampling is then repeated for the same site, eventually resulting in the physical incorporation of the correct nucleotide. However, the premature release can be misread as an incorporation event by a readout system during, e.g., single molecule sequencing, e.g., where the system monitors residence time of the nucleotide analog at the active site as a proxy for incorporation; this can result in sequence read errors which include a nucleotide “insertion” relative to the correct sequence. This phenomenon is termed “branching” and can generate high error rates in single molecule sequencing, especially when chemically modified nucleotides or nucleotide analogs are used.

[0180] Among other aspects, the invention provides methods for generating recombinant polymerases that comprise modifications that reduce the frequency of branching, which can be useful in any number of applications where accuracy of polymerization is beneficial, e.g., high-throughput sequencing systems, e.g., in a zero-mode waveguide (ZMW), SNP genotyping using single base extension methods, real time monitoring of amplification, e.g., RT-PCR methods, and the like. Also provided are compositions that include such polymerases and methods in which these polymerases can //be useful in, e.g., sequencing or making a DNA. In some embodiments, the compositions can also include a nucleotide analog, e.g., a phosphate-labeled nucleotide analog, an analog labeled with a fluorophore, and/or a nucleotide analog comprising from 3-7 phosphate groups, which can be incorporated into a copy nucleic acid by the modified polymerase in response to a DNA template. In some embodiments, the compositions can be present in a sequencing system, e.g. in a zero-mode waveguide, where a polymerase of the invention can optionally be immobilized on a surface.

[0181] Modification of a polymerase, e.g., any of the polymerases described herein, or polymerases homologous to those described herein, by any one or more of the strategies described herein can lower the frequency of these events by creating a more tightly structured binding pocket for non-native nucleotides. For example, any of the mutations described herein for reducing the branching fraction of a polymerase can be present in, or added to, e.g., any of the polymerases of Tables 2, 3, and 4, to modulate the branching fractions of such polymerases. The modified polymerases can include, e.g., L253A, T368F, E375Y, A484E, and/or K512Y substitutions or other mutations described herein. In one embodiment, a polymerase that exhibits a reduced branching frequency can comprise at least one mutation that provides other useful features such as reduced exonuclease activity (e.g., N62D, D12A, D66A, and/or T151 substitutions relative to a wild-type Φ29 polymerase).

[0182] A number of specific examples of a modified polymerase, e.g. modified to lower the frequency of branching events, are described herein. The binding pocket is a portion of the polymerase that encompasses the nucleotide binding site and analog base during the pairing of a nucleotide analog with a template DNA. Because of the physical proximity of the binding pocket to the incoming nucleotide or nucleotide analog, mutations to this region can affect the branching fraction. However, mutations that lower the branching fraction are not limited to this area of the polymerase. For example, relative to a wild-type Φ29 DNA polymerase, these modifications, in addition to those described above, can include any one of, or any combination of: an amino acid substitution at position 153, an amino acid substitution at amino acid position 191, an amino acid substitution at position 388, an amino acid substitution at position 422, an amino acid substitution at position 128; an amino acid substitution at position 253; an amino acid substitution at position 504; an amino acid substitution at position 143; an amino acid substitution at position 183; an amino acid substitution at position 236; an amino acid substitution at position 363; an amino acid substitution at position 215; an amino acid substitution at position 43; an amino acid substitution at position 159; and/or any of the following mutations or combinations thereof: S215D; S43D; T159D; P153L; G191A; T368F; T368P; T368S; T368V; T368N; T368A; T373N; T373V; T373C; I378V; I378F; K379S; K379A; S388A; S388T; K422R; F128M; F128V; I504V; K143D; K512R; Q183S; R236N; L253A; F363Y; L253A, F363Y, and L480M; T368F, K379S, E375Y, and K512Y; T368F and K379S; T368G and K379S; T368F and T373A; E375Y, K512Y and K379S; E375Y, K512Y and T368F; T368F and V514K; T368F and K379T; S388A and P153L; E375Y, K512Y and T368G; T368G and T373A; E375W and T368G; I378K and K379S; T368F and I378K; T368G and I378K; T368G and V514K; E375W and K379T; T373A and K379S; E375W and T373A; E375Y, K512Y and T373A; E375W and I378K; E375Y, K512Y and I378K; T373A and V514K; T373A and I378K; E375Y, K512Y and K379T; I378K and V514K; E375W and V514K; T368G and K379T; and E375Y, K512Y and V514K. A list of specific useful Φ29 mutants and the corresponding reduced branching fraction that they exhibit is provided in Table 5 below. Characteristics of additional useful Φ29 mutants are provided in Table 6. Any of the mutations described herein for reducing the branching fraction of a polymerase can be present in, or added to, e.g., any of the polymerases of Tables 2, 3, and 4, to modulate the branching fractions of such polymerases. Moreover, the mutations provided in Tables 2-4, alone or in combination, can be added to polymerases that include mutations that decrease branching fractions described herein. For comparison, wild-type Φ29 polymerase exhibits a branching fraction of about ≧40% for, e.g., an A488dA4P nucleotide analog. Values in the tables were determined as described in Example 1.

[0183]

	TABLE 5
		Branching
	Mutation Name	Fraction (%)
	N62D_T368F_K379S_E375Y_K512Y	8.01
	N62D_T368F_K379S	6.47
	N62D_T368P	6.58
	N62D_T368G_K379S	6.96
	N62D_T368F_T373A	6.99
	N62D_T368S	7.32
	N62D_E375Y_K512Y_K379S	7.66
	N62D_E375Y_K512Y_T368F	8.53
	N62D_T368F_V514K	8.58
	N62D_T368F_K379T	8.71
	N62D_S388A_P153L	8.93
	N62D_T368V	9.94
	N62D_E375Y_K512Y_T368G	10.14
	N62D_T368D	10.41
	N62D_T368G_T373A	10.69
	N62D_T368N	10.73
	N62D_E375W_T368G	12.04
	N62D_G191A	12.32
	N62D_I378K_K379S	12.47
	N62D_K379A	12.75
	N62D_T368F_I378K	13.30
	N62D_K379S	13.34
	N62D_T368F	13.55
	N62D_T368G_I378K	13.59
	N62D_T368G_V514K	14.01
	N62D_E375W_K379T	14.66
	N62D_T373A_K379S	14.72
	N62D_S388T	14.82
	N62D_E375W_T373A	16.40
	N62D_T368A	16.60
	N62D_I378V	17.38
	N62D_E375Y_K512Y_T373A	17.54
	N62D_T373N	17.63
	N62D_E375W_I378K	17.70
	N62D_E375Y_K512Y_I378K	17.83
	N62D_T373A_V514K	17.87
	N62D_T373A_I378K	17.89
	N62D_T373V	18.26
	N62D_I378F	18.32
	N62D_E375Y_K512Y_K379T	18.72
	N62D_I378K_V514K	18.74
	N62D_T368E	18.82
	N62D_E375W_V514K	19.68
	N62D_T368G_K379T	19.77
	N62D_T373C	20.54
	N62D_K422R	20.68
	N62D_T368G	21.48
	E375Y_K512Y_V514K	24.90
	N62D_F128M	8.86
	N62D_F128V	8.35
	L253A_F363Y_L480M	9.59
	N62D_I504V	5.24
	N62D_K143D	9.66
	N62D_K512R	8.63
	N62D_Q183S	9.62
	N62D_R236N	9.71

[0184]

TABLE 6
Characterization of modified recombinant polymerases including S215D,
S43D, and T159D substitutions.
	Vmax	Km	kcat	specificity
Mutation	(RFU/sec)	(μM)	(bpm)	(kcat/km)	BF %a
N62D_S215D_T368F_E375Y_K512Y	15621.43	2.62	150.62	57.41	6.41
S43D_N62D_T368F_E375Y_K512Y	14682.09	3.53	141.56	40.13	10.01
N62D_T159D_T368F_E375Y_K512Y	13408.60	2.21	129.28	58.59	9.51
N62D_T368F_E375Y_K512Y	13343.50	2.00	128.65	64.18	11.09

[0185] As noted, the branching fraction, e.g., % branching, is a relative measure of the number of times a correctly paired base, e.g., a Watson-Crick paired base, leaves the active site of the polymerase without forming a phosphodiester bond with the 3′OH of the primer-terminus relative to the total number of interactions that occur between the nucleotide (or nucleotide analog) and the binding pocket of the polymerase, e.g., the total number of opportunities the nucleotide or nucleotide analog, e.g., A488dA4P in FIG. 3, has to correctly pair and incorporate. Branching is expressed as a percentage of the dissociation events vs. the total sum events, e.g., dissociation and association events. For example, for an N62D/T368G Φ29 mutant polymerase, for every 100 times an A488dA4P analog interacts with the binding pocket of this polymerase, 21.477 of the events are non-productive dissociation events, e.g., wherein the analog dissociates from the polymerase instead of participating in a polymerization reaction.

[0186] The branching fraction is measured by “loading” a polymerase active site with a cognate-matching nucleotide analog that can bind in the +1 and +2 positions. In the absence of divalent cation this nucleotide cannot be incorporated into the DNA strand, so will pair with the template nucleotide at the +1 position but be released at some frequency specific for that analog/polymerase combination, e.g., the branching rate. This ‘loading’ reaction is then followed by a ‘chase’ reaction consisting of a divalent cation that supports extension, e.g., Mn²⁺), and a terminating-type nucleotide analog, e.g., a dideoxynucleotide, comprising the same base as the cognate-matching analog in the loading step.

[0187] The dideoxy-analog will be incorporated into any +1 sites that are unoccupied and, once added, preclude further extension. Hence polymerase active sites that are already occupied by a paired analog base extend to the +2 position, while those that are not occupied (i.e. “branched”) incorporate the dideoxy-type analog at +1 and do not extend, resulting in a single base addition. The extension products of this reaction are visualized by standard separation methods, e.g., gel or capillary electrophoresis, and the ratio of terminated product that is generated when a dideoxynucleotide is incorporated at the +1 position divided by the total terminated product, e.g., when a dideoxynucleotide is incorporated at both the +1 and +2 positions, indicates the fraction of ‘branched’ events that occur.

[0188] The branching fraction exhibited by a modified polymerase, e.g., a modified Φ29 polymerase, a modified Φ29-type polymerase, or a modified exonuclease-deficient Φ29 polymerase, can be less than a branching fraction exhibited by the parental polymerase for a given nucleotide analog or, e.g., less than 25% for a phosphate-labeled nucleotide analog, less than 20% for the phosphate-labeled analog, less than 15% for the phosphate-labeled analog, or less than 10% for the phosphate-labeled analog.

[0189] In some embodiments, the modified polymerase that exhibits a reduced frequency of branching can also exhibit a K_m for a given phosphate-labeled nucleotide analog, e.g., any of the phosphate-labeled nucleotide analogs described herein, that is less than 10 μM. For enzymes obeying simple Michaelis-Menten kinetics, kinetic parameters are readily derived from rates of catalysis measured at different substrate concentrations. The Michaelis-Menten equation, V=V_max[S]([S]+K_m)⁻¹ relates the concentration of uncombined substrate ([S], approximated by the total substrate concentration), the maximal rate (V_max, attained when the enzyme is saturated with substrate), and the Michaelis constant (K_m, equal to the substrate concentration at which the reaction rate is half of its maximal value), to the reaction rate (V). To determine a K_m for a particular analog a series of extension reactions are performed with a varying concentration of the analog of interest with a fixed, saturating concentration of native nucleotides. A fit of the rate versus the substrate concentration generates estimation of the −K_m as the slope of this line. Modified polymerases that exhibit reductions in branching fraction can also exhibit increased accuracy of nucleotide incorporation. The modified polymerases optionally exhibit improved specificity, e.g., as assessed by determining k_cat/K_m.

[0190] It will be understood that any of the above mutations that decrease the branching fraction of a polymerase can be combined with, e.g., the mutation or mutation combinations provided in Tables 2-4, the mutations described herein that increase closed complex stability, and/or the mutations described herein that reduce reaction rates at one or more steps of the polymerase kinetic cycle.

Modified Recombinant Polymerases with Slow Steps

[0191] The invention also features recombinant polymerases with modifications that decrease the rate of one or more steps within the catalytic cycle, for example, to achieve a reaction system having two kinetically observable reaction steps within an observable phase of the polymerase reaction. Such systems can be useful for observing the activity of a polymerase enzyme in real time, for example, for carrying out single molecule nucleic acid sequencing. For example, a system in which the reaction kinetics exhibit two slow steps within an observable phase can result in more observable sequencing events, allowing for a more accurate determination of a nucleic acid sequence.

[0192] In single molecule DNA sequencing by synthesis, for example as described in Eid et al. (2009) Science 323(5910):133-138, the incorporation of specific nucleotides can be determined by observing bright phases and dark phases which correspond, for example, to reaction steps in which a fluorescent label is associated with the polymerase enzyme, and steps in which the fluorescent label is not associated with the enzyme. In some embodiments of the invention, the polymerase reaction system will exhibit two sequential slow (kinetically observable) reaction steps wherein each of the steps is in a bright phase. In some embodiments of the invention, the system will exhibit two sequential slow reaction steps wherein each of the steps is in a dark phase. In some embodiments, the system will have four slow reaction steps, two slow steps in a bright phase and two slow steps in a dark phase. In some cases, the two or more slow steps are consecutive. In some cases, there can be intervening fast steps between the two or more slow steps.

[0193] An observable phase will generally have a time period during which it is observable. The time period for a bright phase, for example, can be represented by the pulse width. The time period for a dark phase can be represented, for example, by the interpulse distance. The length of each time period will not be the same for each nucleotide addition, resulting in a distribution of the length of the time periods. In some cases, the time periods with the shortest length will not be detected, leading to errors in single molecule sequencing. By designing polymerase reaction systems in which there are two slow, or kinetically observable, steps within an observable phase, the relative number of short, unobservable, time periods can be reduced, resulting in a higher proportion of observable sequencing events and allowing for a more accurate determination of nucleotide sequence. For example, having two slow steps within a bright phase can reduce the incidence of very short pulses, while having two slow steps within a dark phase can reduce the incidence of very short interpulse distances (which occasionally cause pulse merging).

[0194] The modified recombinant polymerases with decreased reaction rates described hereinbelow are desirably employed to obtain such a system with two (or more) slow reaction steps. Optionally, the polymerase reaction conditions, including the type and levels of cofactors, and/or the reaction substrates are also manipulated to achieve such a system, as described in U.S. patent application Ser. No. 12/414,191 filed Mar. 30, 2009, and entitled “Two slow-step polymerase enzyme systems and methods.”

[0195] Polymerase Mediated Synthesis

[0196] In natural polymerase mediated nucleic acid synthesis, a complex is formed between a polymerase enzyme, a template nucleic acid sequence, and a priming sequence that serves as the point of initiation of the synthetic process. During synthesis, the polymerase samples nucleotide monomers from the reaction mix to determine their complementarity to the next base in the template sequence. When the sampled base is complementary to the next base, it is incorporated into the growing nascent strand. This process continues along the length of the template sequence to effectively duplicate that template. Although described in a simplified schematic fashion, the actual biochemical process of incorporation is relatively complex.

[0197] The process can be described as a sequence of steps, wherein each step can be characterized as having a particular forward and reverse reaction rate that can be represented by a rate constant. One representation of the incorporation biochemistry is provided in FIG. 12. It is to be understood that the scheme shown in FIG. 12 does not provide a unique representation of the process. In some cases, the process can be described using fewer steps. For example, the process is sometimes represented without inclusion of the enzyme isomerization steps 106 and 110. Alternatively, the process can be represented by including additional steps such as cofactor binding. Generally, steps which can be slow, and thus limit the rate of reaction, will tend to be included. Various schemes can be used to represent a polymerization reaction, e.g., having one or two slow steps, that may have more or fewer identified steps.

[0198] As shown in FIG. 12, the synthesis process begins with the binding of the primed nucleic acid template (D) to the polymerase (P) at step 102. Nucleotide (N) binding with the complex occurs at step 104. Step 106 represents the isomerization of the polymerase from the open to closed configuration. Step 108 is the chemistry step where the nucleotide is incorporated into the growing strand of the nucleic acid being synthesized. At step 110, polymerase isomerization occurs from the closed to the open position. The polyphosphate component that is cleaved upon incorporation is released from the complex at step 112. The polymerase then translocates on the template at step 114. As shown, the various steps can include reversible paths and may be characterized by the reaction constants shown in FIG. 12 where:

• [0199] k_on/k_off=DNA binding/release;
• [0200] k₁/k₋₁=nucleotide binding/release;
• [0201] k₂/k₋₂=polymerase isomerization (open/closed);
• [0202] k₃/k₋₃=nucleotide incorporation (chemistry);
• [0203] k₄/k₋₄=polymerase isomerization (closed/open);
• [0204] k₅/k₋₅=polyphosphate release/binding;
• [0205] k₆/k₋₆=polymerase translocation.

[0206] Thus, during steps 104 through 110, the nucleotide is retained within the overall complex, and during steps 104 and 106, reversal of the reaction step will yield an unproductive event, i.e., not resulting in incorporation. For example, a bound nucleotide at step 104 may be released regardless of whether it is the correct nucleotide for incorporation.

[0207] By selecting the appropriate polymerase enzyme, polymerase reaction conditions, and polymerase substrates, the absolute and relative rates of the various steps can be controlled. Controlling the reaction such that the reaction exhibits two or more sequential kinetically observable, or slow, steps can produce a nucleic acid polymerization reaction in which the incorporation of the nucleotides can be observed more accurately. These characteristics are particularly useful for sequencing applications, and in particular single molecule DNA sequencing.

[0208] In some cases, the invention involves a process having two or more slow steps that comprise steps after nucleotide binding through the step of product release. For the mechanism shown in FIG. 12, this would be, for example, any of steps 106, 108, 110, and 112. In some cases, steps 108 (nucleotide incorporation) and 112 (product release) are the two slow steps. In some cases, the invention involves a process having two or more slow steps that comprise the steps after product release through nucleotide binding. For the mechanism shown in FIG. 12, this would include steps 114 and 104.

[0209] In some cases, the invention involves a process in which there are two or more slow steps in two different observable phases within the polymerization, for example, two slow steps in a bright phase and two slow steps in a dark phase. For example, this could include a system having two slow steps in the steps after nucleotide binding through product release, and two slow steps for the steps after product release through nucleotide binding. As is described herein, producing a process in which there are two slow steps in these portions of the polymerase reaction can result in a higher proportion of detectable enzyme states which can be useful, for example, to observe the sequential incorporation of nucleotides for nucleotide sequencing.

[0210] By the term slow step is generally meant a kinetically observable step. An enzymatic process, such as nucleic acid polymerization, can have both slower, kinetically observable steps and faster steps which are so fast that they have no measurable effect on the kinetics, or rate, of the reaction. In some reactions, there can be a single rate limiting step. For such reactions, the kinetics can be characterized by the rate of that single step. Other reactions will not have a single rate limiting step, but will have two or more steps which are close enough in rate such that the characteristics of each will contribute to the kinetics of the reaction. For the current invention, the slow, or kinetically observable, steps need not be the slowest step or the rate limiting step of the reaction. For example, a process of the current invention can involve a reaction in which step 104, nucleotide addition, is the slowest (rate limiting) step, while two or more of steps 106, 108, 110, or 112 are each kinetically observable.

[0211] As used herein, the term rate as applied to the steps of a reaction can refer to the average rate of reaction. For example, when observing a single molecule reaction, there will generally be variations in the rates as each individual nucleotide is added to a growing nucleic acid. In such cases the rate of the reaction can be represented by observing a number of individual events, and combining the rates, for example, by obtaining an average of the rates.

[0212] As used herein, the reference to the rate of a step or rate constant for a step can refer to the forward reaction rate of the polymerase reaction. As is generally understood in the art, reaction steps can be characterized as having forward and reverse rate constants. For example, for step 108, k₃ represents the forward rate constant, and k₋₃ represents the reverse rate constant for the nucleotide incorporation. Some reaction steps, such as step 108, constitute steps which would be expected to be first order steps. Other steps, such as the forward reaction of step 104, with rate constant k₂, would be expected to be second order rate constants. For the purposes of the invention, for comparing the rate or the rate constant of a first order to a second order step, the second order rate constant k₂ can be treated as a pseudo-first order rate constant with the value [N]*k₂ where the concentration of nucleotide [N] is known.

[0213] For some applications, it is desirable that the kinetically observable steps of the invention have rate constants that are lower than about 100 per second. In some cases, the rate constants are lower than about 60 per second, lower than about 50 per second, lower than about 30 per second, lower than about 20 per second, lower than about 10 per second, lower than about 5 per second, lower than about 2 per second, or lower than about 1 per second.

[0214] In some embodiments the slowest of the two or more kinetically observable steps has a rate constant when measured under single molecule conditions of between about 60 to about 0.5 per second, about 30 per second to about 2 per second, or about 10 to about 3 per second.

[0215] The ratio of the rate constants of each the two or more slow steps is generally greater than 1:10; in some cases the ratio of the rate constants is about 1:5, in some cases the ratio of the rate constants is about 1:2, and in some cases, the ratio of rate constants is about 1:1. The ratio of the rate constants can be between about 1:10 and about 1:1, between about 1:5 and about 1:1, or between about 1:2 and about 1:1.

[0216] In some cases it is useful to consider the two slow-step system in terms of rates rather than rate constants. It is generally desirable that the kinetically observable steps of the invention have rates that are lower than about 100 molecules per second when the reactions are carried out under single-molecule conditions. In some cases, the rates are lower than about 60 molecules per second, lower than about 50 molecules per second, lower than about 30 molecules per second, lower than about 20 molecules per second, lower than about 10 molecules per second, lower than about 5 molecules per second, lower than about 2 molecules per second, or lower than about 1 molecule per second.

[0217] In some embodiments the slowest of the two or more kinetically observable steps has a rate when measured under single molecule conditions of between about 60 to about 0.5 molecules per second, about 30 molecules per second to about 2 molecules per second, or about 10 to about 3 molecules per second.

[0218] The ratio of the rates of each the two or more slow steps is generally greater than 1:10. In some cases the ratio of the rates is about 1:5, in some cases the ratio of the rates is about 1:2, and in some cases, the ratio of rates is about 1:1. The ratio can be between about 1:10 and about 1:1, between about 1:5 and about 1:1, or between about 1:2 and about 1:1.

[0219] Any one (or more) of the steps described above is optionally slowed in the recombinant polymerases of the invention, e.g., to produce a polymerase useful in achieving a reaction system exhibiting two slow steps.

[0220] Sequencing by Incorporation

[0221] For sequencing processes that rely upon monitoring of the incorporation of nucleotides into growing nascent strands being synthesized by the complex, the progress of the reaction through these steps is of significant importance. In particular, for certain “real time” nucleotide incorporation monitoring processes, the detectability of the incorporation event is improved based upon the amount of time the nucleotide is incorporated into and retained within the synthesis complex during its ultimate incorporation into a primer extension product.

[0222] By way of example, in certain exemplary processes, the presence of the nucleotide in the synthesis complex is detected either by virtue of a focused observation of the synthesis complex, or through the use of interactive labeling techniques that produce characteristic signals when the nucleotide is within the synthesis complex. See, e.g., Levene et al. (2003) Science 299:682-686 and Eid et al. (2009) Science 323(5910):133-138, the full disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0223] In a first exemplary technique, as schematically illustrated in FIG. 13 Panel A, a nucleic acid synthesis complex, including a polymerase enzyme 202, a template sequence 204 and a complementary primer sequence 206, is provided immobilized within an observation region 200, that permits illumination (as shown by hv) and observation of a small volume that includes the complex without excessive illumination of the surrounding volume (as illustrated by dashed line 208). By illuminating and observing only the volume immediately surrounding the complex, one can readily identify fluorescently labeled nucleotides that become incorporated during that synthesis, as such nucleotides are retained within that observation volume by the polymerase for longer periods than those nucleotides that are simply randomly diffusing into and out of that volume.

[0224] In particular, as shown in Panel B of FIG. 13, when a nucleotide, e.g., A, is incorporated into by the polymerase, it is retained within the observation volume for a prolonged period of time, and upon continued illumination yields a prolonged fluorescent signal (shown by peak 210). By comparison, randomly diffusing and not incorporated nucleotides remain within the observation volume for much shorter periods of time, and thus produce only transient signals (such as peak 212), many of which go undetected, due to their extremely short duration.

[0225] In particularly preferred exemplary systems, the confined illumination volume is provided through the use of arrays of optically confined apertures termed zero mode waveguides (ZMWs), e.g., as shown by confined reaction region 200 (see, e.g., U.S. Pat. No. 6,917,726, which is incorporated herein by reference in its entirety for all purposes). For sequencing applications, the DNA polymerase is typically provided immobilized upon the bottom of the ZMW. See, e.g., Korlach et al. (2008) PNAS U.S.A. 105(4):1176-1181, which is incorporated herein by reference in its entirety for all purposes.

[0226] In operation, the fluorescently labeled nucleotides (shown as A, C, G and T) bear one or more fluorescent dye groups on a terminal phosphate moiety that is cleaved from the nucleotide upon incorporation. As a result, synthesized nucleic acids do not bear the build-up of fluorescent labels, as the labeled polyphosphate groups diffuses away from the complex following incorporation of the associated nucleotide, nor do such labels interfere with the incorporation event. See, e.g., Korlach et al., Nucleosides, Nucleotides and Nucleic Acids, 27:1072:1083, 2008.

[0227] In a second exemplary technique, the immobilized complex and the nucleotides to be incorporated are each provided with interactive labeling components. Upon incorporation, the nucleotide borne labeling component is brought into sufficient proximity to the complex borne (or complex proximal) labeling component, such that these components produce a characteristic signal event. For example, the polymerase may be provided with a fluorophore that provides fluorescent resonant energy transfer (FRET) to appropriate acceptor fluorophores. These acceptor fluorophores are provided upon the nucleotide to be incorporated, where each type of nucleotide bears a different acceptor fluorophore, e.g., that provides a different fluorescent signal. Upon incorporation, the donor and acceptor are brought close enough together to generate energy transfer signal. By providing different acceptor labels on the different types of nucleotides, one obtains a characteristic FRET-based fluorescent signal for the incorporation of each type of nucleotide, as the incorporation is occurring.

[0228] In a related aspect, a nucleotide analog may include two interacting fluorophores that operate as a donor/quencher pair, where one member is present on the nucleobase or other retained portion of the nucleotide, while the other member is present on a phosphate group or other portion of the nucleotide that is released upon incorporation, e.g., a terminal phosphate group. Prior to incorporation, the donor and quencher are sufficiently proximal on the same analog as to provide characteristic signal quenching. Upon incorporation and cleavage of the terminal phosphate groups, e.g., bearing a donor fluorophore, the quenching is removed and the resulting characteristic fluorescent signal of the donor is observable.

[0229] In exploiting the foregoing processes, where the incorporation reaction occurs too rapidly, it may result in the incorporation event not being detected, i.e., the event speed exceeds the detection speed of the monitoring system. The missed detection of incorporated nucleotides can lead to an increased rate of errors in sequence determination, as omissions in the real sequence. In order to mitigate the potential for missed pulses due to short reaction times, in one aspect, the current invention can result in increased reaction time for incorporations. An advantage of employing polymerases with reduced reaction rates, e.g., polymerases exhibiting decreased rates and/or two slow-step kinetics, is an increased frequency of longer, detectable, binding events. This advantage may also be seen as an increased ratio of longer, detectable pulses to shorter, non-detectable pulses, where the pulses represent binding events.

[0230] Single molecule sequencing often involves the optical observation of the polymerase process during the process of nucleotide incorporation, for example, observation of the enzyme-DNA complex. During this process, there are generally two or more observable phases. For example, where a terminal-phosphate labeled nucleotide is used and the enzyme-DNA complex is observed, there is a bright phase during the steps where the label is incorporated with (bound to) the polymerase enzyme, and a dark phase where the label is not incorporated with the enzyme. For the purposes of this invention, both the dark phase and the bright phase are generally referred to as observable phases, because the characteristics of these phases can be observed.

[0231] Whether a phase of the polymerase reaction is bright or dark can depend, for example, upon how and where the components of the reaction are labeled and also upon how the reaction is observed. For example, as described above, the phase of the polymerase reaction where the nucleotide is bound can be bright where the nucleotide is labeled on its terminal phosphate. However, where there is a quenching dye associated with the enzyme or template, the bound state may be quenched, and therefore be a dark phase. Analogously, in a ZMW, the release of the terminal phosphate may result in a dark phase, whereas in other systems, the release of the terminal phosphate may be observable, and therefore constitute a bright phase.

[0232] For example, consider again the reaction scheme of FIG. 12 in the context of the sequencing by incorporation embodiment described above which utilizes nucleotides having labels on their terminal phosphates. For this system, intermediates PDN, P*DN, P*D₊₁PP_i, and PD₊₁PP_i would all represent bright states of a bright phase because for each of these intermediates, the label is associated with the polymerase enzyme. In contrast, intermediates PD₊₁ and PD correspond to dark states of a dark phase, because for these intermediates, no dye is associated with the polymerase enzyme. In one aspect of the invention, any step (and preferably any two of the steps) which proceed from a bright intermediate, e.g. steps 106, 108, 110, and 112 of FIG. 12 are slow. By having two or more sequential bright steps that are slow, the relative number of longer pulses and detectable incorporation events increases.

[0233] Another example of a polymerase reaction with distinct observable phases is one in which the nucleotide is labeled such that its label does not dissociate from the enzyme upon product release, for example where the nucleotide is labeled on the base or on the sugar moiety. Here, the phase in which the label is associated with the active site of the enzyme (bright or dark) may extend past product release until translocation. For this example, an observable phase may extend from nucleotide binding until translocation.

[0234] In addition, the systems of the present invention may have two or more different distinct bright phases, for example, phases that can be distinguished based on different colors, e.g. different fluorescent emission wavelengths in the different observable phases. For all of these cases, it can be advantageous to have more than one rate limiting (kinetically observable) step within a phase. Having more than one rate limiting step within a phase can result in a distribution of pulse widths having relatively fewer undetectable or poorly detectable short pulses.

[0235] While not being bound by theory, the following theoretical basis is provided for obtaining improved single molecule sequencing results by using a system having two or more slow steps. A model for the effect of two slow steps on the probability density for residence time is described herein. FIG. 14 shows a plot of calculated probability density for residence time for cases in which (1) one step is rate limiting and (2) two equivalent rate limiting (slow) steps are present for the observable phase in which the nucleotide is associated with the enzyme.

[0236] For the case in which one step is rate limiting, the probability distribution for the binding time can be represented by the single exponential equation:
y=A₀e^−kt  Eq. 1

[0237] This represents the case in which, for example, incorporation of nucleotide into the growing nucleic acid (step 108 in FIG. 12) is the single slow step.

[0238] FIG. 14 illustrates that where one slow-step is present in this phase, there is an exponentially decreasing probability of a given residence time as the residence time increases, providing a distribution in which there is a relatively high probability that the residence time will be short.

[0239] For the case in which there are two slow steps in this phase, for example where both the incorporation step (step 108 in FIG. 12) and the release of product (PPi) step (step 112 in FIG. 12) are slow, the probability density versus residence time can be represented by a double exponential equation:
y=A₀e^−k1t−B₀e^−k2t  Eq. 2

[0240] FIG. 14 illustrates that for the case in where there are two slow steps, the probability of very fast residence times is relatively low as compared to the case having one slow step. In addition, the probability distribution for two slow steps exhibits a peak in the plot of probability density versus residence time. This type of residence time distribution can be advantageous for single molecule sequencing where it is desired to measure a high proportion of binding events and where fast binding events may be unreliably detected.

[0241] Typically, for a given illumination/detection system there will be a minimum detection time below which events, such as binding events, will be unreliably detected or not detected at all. This minimum detection time can be attributed, for example, to the frame acquisition time or frame rate of the optical detector, for example, a CCD camera. A discussion of detection times and approaches to detection for these types of systems is provided in U.S. patent application Ser. No. 12/351,173 the full disclosures of which are incorporated herein by reference in their entirety for all purposes. FIG. 14 includes a line which indicates a point where the residence time equals a minimum detection time (Tmin). The area under the curve in the region below Tmin represents the population of short pulses which will not be accurately detected for this system. It can be seen from FIG. 14 that the relative proportion of binding times that fall below Tmin is significantly lower for the case in which the reaction exhibits two sequential slow steps as compared to the case where the reaction exhibits one slow step.

[0242] Thus, as described above, one aspect of the invention relates to methods, systems, and compositions for performing nucleic acid sequencing with a nucleic acid synthesis reaction in which the reaction exhibits two or more slow steps within a bright phase, e.g., employing a modified polymerase exhibiting one or more slowed step. In addition, an aspect of the invention relates to nucleic acid synthesis reactions having two or more slow states wherein each of the slow steps proceeds from a state in which the labeled component is associated with the polymerase enzyme.

[0243] In some embodiments of the invention, the two or more slow steps are within a dark phase. In some cases the two or more slow steps proceed from states in which the labeled component is not associated with the enzyme. Having two or more slow states that proceed from a dark intermediate can be advantageous, for example, for lowering the frequency of events having a very short dark state or having a very short interpulse distance. The advantage of this type of system can be demonstrated by again considering FIG. 12 in the context of the sequencing by incorporation embodiment described above which utilizes nucleotides having labels on their terminal phosphates. In this system, intermediates PD₊₁ and PD can correspond to dark states within a dark phase, for example in a ZMW, because for these intermediates, no dye is associated with the polymerase enzyme.

[0244] The steps that comprise the two slow steps can include, for example, nucleotide addition, enzymatic isomerization such as to or from a closed state, cofactor binding or release, product release, incorporation of nucleic acid into the growing nucleic acid, or translocation. As noted, one or more of the slow steps can be achieved by modification of the polymerase. Various exemplary modified recombinant polymerases exhibiting one or more slow steps are described herein, along with strategies for producing additional such polymerases.

[0245] Modified Recombinant Polymerases Exhibiting Slow Steps

[0246] The invention features recombinant polymerases with modifications that slow one or more steps in the catalytic cycle, for example, to achieve two limiting steps as described above. Accordingly, one aspect of the invention provides a modified recombinant DNA polymerase that comprises one or more mutations relative to a parental polymerase and that exhibits a first rate constant for a first step in its catalytic cycle that is less than a first rate constant for the first step exhibited by the parental polymerase. For example, the first rate constant exhibited by the modified recombinant polymerase can be less than 0.5 times, less than 0.25 times, or even less than 0.1 times the first rate constant exhibited by the parental polymerase.

[0247] As noted above, to achieve a two slow step enzyme it is typically desirable to decrease the rate of a step which is not already rate limiting. Thus, in one aspect, the first step is not rate limiting in the catalytic cycle of the parental polymerase. Also as noted above, polymerases exhibiting approximately the same rate for two sequential (though not necessarily consecutive) steps are desirable. Thus, the modified recombinant polymerase optionally exhibits a second rate constant for a second step in its catalytic cycle, where the second rate constant is between 0.1 and 10 times the first rate constant. Preferably, the second rate constant exhibited by the modified recombinant polymerase is between 0.2 and 5 times the first rate constant exhibited by the modified recombinant polymerase. More preferably, the second rate constant exhibited by the modified recombinant polymerase is approximately equal to the first rate constant exhibited by the modified recombinant polymerase (e.g., within 10%, 5%, or 1%). In one exemplary embodiment, the second step involves incorporation of a bound nucleotide or nucleotide analog, the first step involves release of a polyphosphate product, and the second rate constant exhibited by the modified recombinant polymerase is between 0.2 and 1 times the first rate constant exhibited by the modified recombinant polymerase. In another exemplary embodiment, the first step involves translocation and the second step involves nucleotide or analog binding. It will be understood that in this context, the terms first step and second step are merely used for convenience in referring to two different steps and do not imply any particular order of occurrence (that is, the first step can precede or follow the second and need not be the initial event in the catalytic pathway).

[0248] Optionally, the second step is rate limiting in the catalytic cycle of the parental polymerase. The first or second step can be rate limiting in the catalytic cycle of the modified polymerase. As another option, however, the first and/or second steps are not rate limiting for the catalytic cycle, but are limiting for a portion of the cycle (e.g., the bright or dark portion). Optionally, the polymerase exhibits two limiting steps in the bright portion of the cycle and two in the dark portion.

[0249] Since for many polymerases nucleotide incorporation is rate limiting, the second step can, for example, involve incorporation of a bound nucleotide or nucleotide analog, e.g., an analog having from 3-7 phosphate groups, e.g., with a terminal label. The second rate constant would then be k₃ according to the catalytic cycle illustrated in FIG. 12.

[0250] Essentially any step in the cycle can correspond to the first step whose rate is slowed, for example, nucleotide or analog binding, translocation, isomerization, e.g., of the polymerase or analog, chemistry (incorporation or transphosphorylation), pre-product release isomerization, and product release. Optionally, an extra kinetic step is created that does not occur in the parental enzyme's cycle. In one exemplary class of useful embodiments, the first step involves release of a polyphosphate product, including, for example (and depending on the type of nucleotide or analog incorporated), a pyrophosphate, a polyphosphate with three or more phosphate groups, a labeled polyphosphate, etc. Polyphosphate release is typically so fast as to be undetectable by routine techniques, but in the polymerases of the invention release can be sufficiently slowed as to be observable and permit determination of a rate constant (e.g., k₅). Accordingly, the first rate constant exhibited by a modified recombinant polymerase for release of polyphosphate can be less than 100/second, less than 75/second, or even less than 50/second.

[0251] The parental and modified polymerases can display comparable rates for the second step, or the second step can also be slowed for the recombinant polymerase. Thus, the second rate constant exhibited by the modified recombinant polymerase is optionally smaller than the second rate constant exhibited by the parental polymerase for the second step, e.g., less than 0.5 times, less than 0.25 times, or even less than 0.1 times the second rate constant exhibited by the parental polymerase.

[0252] A modified polymerase (e.g., a modified recombinant Φ29-type DNA polymerase) that exhibits one or more slow steps optionally includes a mutation (e.g., an amino acid substitution or insertion) at one or more of positions 484, 249, 179, 198, 211, 255, 259, 360, 363, 365, 370, 372, 378, 381, 383, 387, 389, 393, 433, 478, 480, 514, 251, 371, 379, 380, 383, 458, 486, 101, 188, 189, 303, 313, 395, 414, 497, 500, 531, 532, 534, 558, 570, 572, 574, 64, 305, 392, 402, 422, 496, 529, 538, 555, 575, 254, 390, 372-397, 507-514, 93, 129, 170, 176, 180, 181, 182, 185, 190, 203, 204, 247, 329, 330, 361, 399, 420, 427, 436, 459, 477, 487, and 567, or any other position where a mutation is noted herein, where numbering of positions is relative to wild-type Φ29 polymerase. For example, relative to wild-type Φ29 a modified recombinant polymerase can include at least one amino acid substitution or combination of substitutions selected from the group consisting of: an amino acid substitution at position 484; an amino acid substitution at position 198; an amino acid substitution at position 381; an amino acid substitution at position 387 and an amino acid substitution at position 484; an amino acid substitution at position 372, an amino acid substitution at position 480, and an amino acid substitution at position 484; an amino acid substitution at position 372, an amino acid substitution at position 387, and an amino acid substitution at position 480; an amino acid substitution at position 372, an amino acid substitution at position 387, and an amino acid substitution at position 484; an amino acid substitution at position 372, an amino acid substitution at position 387, an amino acid substitution at position 478, and an amino acid substitution at position 484; A484E; A484Y; N387L; T372Q; T372Y; T372Y and K478Y; K478Y; I370W; F198W; L381A; T368F; A484E, E375Y, K512Y, and T368F; A484Y, E375Y, K512Y, and T368F; N387L, E375Y, K512Y, and T368F; T372Q, E375Y, K512Y, and T368F; T372L, E375Y, K512Y, and T368F; T372Y, K478Y, E375Y, K512Y, and T368F; I370W, E375Y, K512Y, and T368F; F198W, E375Y, K512Y, and T368F; L381A, E375Y, K512Y, and T368F; and E375Y, K512Y, and T368F. A K512F substitution (or K512W, K512L, K5121, K512V, K512H, etc.) is optionally employed, e.g., where a K512Y substitution is listed herein. As another example, the modified polymerase can include an insertion of at least one amino acid (e.g., 1-7 amino acids, e.g., glycine) within residues 372-397 and/or 507-514. For example, a glycine residue can be introduced after residue 374, 375, 511, and/or 512 (designated as 374.1G, 375.1G, etc.).

[0253] A list of exemplary mutations and combinations thereof is provided in Table 7, and additional exemplary mutations are described herein. Essentially any of these mutations, or any combination thereof, can be introduced into a polymerase to produce a modified recombinant polymerase (e.g., into wild-type Φ29, an exonuclease deficient Φ29-type polymerase, an L253A/E375Y/A484E/K512Y, E375Y/K512Y/T368F, E375Y/K512Y/T368F/A484E, E375Y/K512Y/A484E modified Φ29 polymerase, or a polymerase from any of Tables 2, 3, 4, 6, 9 or 10, as just a few examples).

[0254]

TABLE 7
Mutation	Rationale
D249E	metal coordination
A484E	metal coordination
D249E/A484E	metal coordination
A484D	metal coordination
A484H	metal coordination
A484Y	metal coordination
D249E/A484D	metal coordination
D249E/A484H	metal coordination
D249E/A484Y	metal coordination
374.1G/375.1A	dye interaction
374.1Gins/375.1Gins	dye interaction
V514Y	dye interaction
V514F	dye interaction
511.1G/K512Y/512.1G	dye interaction
T372H	closed conformation of fingers
T372V	closed conformation of fingers
T372I	closed conformation of fingers
T372F	closed conformation of fingers
T372Y	closed conformation of fingers
T372N	closed conformation of fingers
T372Q	closed conformation of fingers
T372L	closed conformation of fingers
T372L/K478Y	closed conformation of fingers
T372Y/K478Y	closed conformation of fingers
T372Y/K478L	closed conformation of fingers
K478Y	closed conformation of fingers
D365N	closed conformation of fingers
D365Q	closed conformation of fingers
L480H	closed conformation of fingers
L480F	closed conformation of fingers
L381A	closed conformation of finger and exo
I179A	closed conformation of finger and exo
I378A	closed conformation of finger and exo
I179A/L381A	closed conformation of finger and exo
I179A/I378A/L381A	closed conformation of finger and exo
I370A/I378A	closed conformation of finger and exo
I179A/I370A/I378A/L381A	closed conformation of finger and exo
I179W	closed conformation of finger and exo
I179H	closed conformation of finger and exo
F211A	closed conformation of finger and exo
F211W	closed conformation of finger and exo
F211H	closed conformation of finger and exo
F198A	closed conformation of finger and exo
F198W	closed conformation of finger and exo
F198H	closed conformation of finger and exo
P255A	closed conformation of finger and exo
P255W	closed conformation of finger and exo
P255H	closed conformation of finger and exo
Y259A	closed conformation of finger and exo
Y259W	closed conformation of finger and exo
Y259H	closed conformation of finger and exo
F360A	closed conformation of finger and exo
F360W	closed conformation of finger and exo
F360H	closed conformation of finger and exo
F363A	closed conformation of finger and exo
F363H	closed conformation of finger and exo
F363W	closed conformation of finger and exo
I370W	closed conformation of finger and exo
I370H	closed conformation of finger and exo
K371A	closed conformation of finger and exo
K371W	closed conformation of finger and exo
I378H	closed conformation of finger and exo
I378W	closed conformation of finger and exo
L381W	closed conformation of finger and exo
L381H	closed conformation of finger and exo
K383N	closed conformation of finger and exo
K383A	closed conformation of finger and exo
L389A	closed conformation of finger and exo
L389W	closed conformation of finger and exo
L389H	closed conformation of finger and exo
F393A	closed conformation of finger and exo
F393W	closed conformation of finger and exo
F393H	closed conformation of finger and exo
I433A	closed conformation of finger and exo
I433W	closed conformation of finger and exo
I433H	closed conformation of finger and exo
K383L	phosphate backbone interaction
K383H	phosphate backbone interaction
K383R	phosphate backbone interaction
Q380R	phosphate backbone interaction
Q380H	phosphate backbone interaction
Q380K	phosphate backbone interaction
K371L	phosphate backbone interaction
K371H	phosphate backbone interaction
K371R	phosphate backbone interaction
K379L	phosphate backbone interaction
K379H	phosphate backbone interaction
K379R	phosphate backbone interaction
E486A	phosphate backbone interaction
E486D	phosphate backbone interaction
N387L	incoming nucleotide base and translocation
N387F	incoming nucleotide base and translocation
N387V	incoming nucleotide base and translocation
N251H	phosphate interaction
N251Q	phosphate interaction
N251D	phosphate interaction
N251E	phosphate interaction
N251K	phosphate interaction
N251R	phosphate interaction
A484K	phosphate interaction
A484R	phosphate interaction
K383Q	phosphate interaction
K383N	phosphate interaction
K383T	phosphate interaction
K383S	phosphate interaction
K383A	phosphate interaction
I179H/I378H	closed conformation
I179W/I378W	closed conformation
I179Y/I378Y	closed conformation
K478L
I378Y
I370A
I179Y
N387L/A484E
N387L/A484Y
T372Q/N387L/A484E
T372Q/N387L/A484Y
T372L/N387L/A484E
T372L/N387L/K478Y/
A484Y
T372Y/N387L/K478Y/
A484E
T372Y/N387L/K478Y/
A484Y