pAVEC

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/473,431, filed Apr. 8, 2011; which is herein incorporated by reference in its entirety.

[0002] The field of the invention relates to plasmids which may be used, for example, to express polypeptides of interest.

[0003] Culturing cells for the commercial production of therapeutic proteins is a costly process. The equipment required is expensive and research and development and production costs are high. Development of cell culture processes which maximize the quantity of therapeutic protein produced per liter of cell culture will minimize the resources necessary to produce a given quantity of the protein. It is, thus, desirable to use commercially viable reagents which produce large quantities of proteins.

[0004] Many naturally occurring cells do not produce large quantities of desired proteins, under standard culture conditions. Rather, extensive research and development of cell culture processes, which coax cells in culture to generate large quantities of therapeutic protein, must be performed. Typically, identifying plasmid vectors useful for expressing a protein at a high level requires a significant amount of inventive input.

[0005] The present invention provides, in part, an isolated pAVEC plasmid vector which is linear or circular comprising a multiple cloning site comprising the restriction sites:

[0006]
e.g., comprising the nucleotide sequence of SEQ ID NO: 2; for example, the plasmid vector is characterized by the plasmid map of FIG. 1; wherein the plasmid comprises the nucleotide sequence set forth in SEQ ID NO: 1. The pAVEC plasmid vectors may be circular and, thus, capable of autonomous ectopic reproduction in a host cell; or linear and capable of use, e.g., for integration into the chromosomal DNA of a host cell.

[0007] The present invention further comprises a method for making a recombined plasmid comprising cleaving a pAVEC plasmid and ligating the ends of the cleaved plasmid to compatible ends of an introduced polynucleotide (e.g., encoding an immunoglobulin such as Abciximab, Adalimumab, Alemtuzumab, Basiliximab, Bevacizumab, Cetuximab, Certolizumab pegol, Dalotuzumab (MK0646), Daclizumab, Denosumab, Eculizumab, Efalizumab, Gemtuzumab, Ibritumomab tiuxetan, Infliximab, Muromonab-CD3, Natalizumab, Omalizumab, Palivizumab, Panitumumab, Ranibizumab, Rituximab, Robatumumab, Tositumomab, ALD518 and Trastuzumab; or, an immunoglobulin chain that is a heavy or light chain of an antibody or antigen-binding fragment thereof that binds specifically to an antigen selected from the group consisting of: VEGF, VEGFR, EGF, EGFR, TNFalpha, TGFbeta, TRAIL-R1, Nav1.7, Nav1.8, ERK, MEK, TRAIL-R2, IL-6, IL-6R, IGF1R, IL-23p19, IL-23R, PCSK9, CD20, RANKL, RANK, CD33, CD11a, ErbB2, IgE, a G-protein coupled receptor (GPCR) an HIV antigen, an HCV antigen and a respiratory syncytial virus (RSV) antigen) such that a closed circular plasmid is produced, e.g., wherein the introduced polynucleotide is operably linked to a promoter in the plasmid, such as the hCMV promoter. Any recombined plasmid produced by such a method forms part of the present invention.

[0008] The present invention further includes an isolated host cell (e.g., bacterial or mammalian cell such as a CHO cell) comprising an isolated host cell comprising any of the pAVEC plasmids of the present invention.

[0009] The present invention also provides a method for producing a recombinant polypeptide in an isolated host cell, comprising introducing the any recombined pAVEC plasmid vector of the present invention that comprises a polynucleotide that encodes a polypeptide e.g., wherein the polynucleotide encoding the polypeptide is operably linked to a promoter in the plasmid, such as the hCMV promoter; into the host cell under conditions which allow for expression of the polypeptide; and optionally, purifying the polypeptide.

[0010] Furthermore, the present invention includes a kit comprising a pAVEC plasmid vector of the invention and one or more additional components.

[0013] The present invention provides an expression vector useful for recombinant protein expression in any cell, for example in a mammalian cell, a bacterial cell, a yeast cell or an insect cell. The vector may be used to transiently or stably express a broad range of recombinant proteins. The multiple cloning site of the vector offers many common and rare restriction sites to accommodate a variety of expression cassettes.

[0014] A pAVEC plasmid is a plasmid characterized by the plasmid map set forth in FIG. 1 and/or comprising or consisting of the nucleotide sequence of SEQ ID NO: 1; the term also encompasses a recombined plasmid produced by a process comprising cleaving pAVEC plasmid and ligating the ends of the cleaved plasmid to compatible ends of an introduced polynucleotide such that a closed circular plasmid is produced which comprises the introduced polynucleotide.

[0015] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook, et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0016] A “polynucleotide,” “nucleic acid” or “nucleic acid molecule” includes DNA or RNA. For example, in an embodiment of the invention, the polynucleotide is the circular plasmid pAVEC.

[0017] A “polynucleotide sequence,” “nucleic acid sequence” or “nucleotide sequence” is a series of nucleotides in a nucleic acid, such as DNA or RNA, and means any chain of two or more nucleotides.

[0018] A “coding sequence” or a sequence “encoding” an expression product such as a RNA or peptide (e.g., an immunoglobulin chain), is a nucleotide sequence that, when expressed, results in production of the product.

[0019] As used herein, the term “oligonucleotide” refers to a nucleic acid, generally of no more than about 300 nucleotides (e.g., 30, 40, 50, 60, 70, 80, 90, 150, 175, 200, 250 or 300), that may be hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides are usually single-stranded, but may be double-stranded. Oligonucleotides can be labeled, e.g., by incorporation of ³²P-nucleotides, ³H-nucleotides, ¹⁴C-nucleotides, ³⁵S-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of the gene, or to detect the presence of nucleic acids. Generally, oligonucleotides are prepared synthetically, e.g., on a nucleic acid synthesizer.

[0020] A “protein sequence,” “peptide sequence” or “polypeptide sequence,” or “amino acid sequence” refers to a series of two or more amino acids in a protein, peptide or polypeptide.

[0021] “Protein,” “peptide” or “polypeptide” includes a contiguous string of two or more amino acids.

[0022] The term “isolated polynucleotide” or “isolated polypeptide” includes a polynucleotide (e.g., RNA or DNA molecule, or a mixed polymer) or a polypeptide, respectively, which is partially or fully separated from other components that are normally found in cells or in recombinant DNA expression systems or any other contaminant. These components include, but are not limited to, cell membranes, cell walls, ribosomes, polymerases, serum components and extraneous genomic sequences.

[0023] An isolated polynucleotide (e.g., pAVEC) or polypeptide will, preferably, be an essentially homogeneous composition of molecules but may contain some heterogeneity.

[0024] The term “host cell” includes any cell of any organism that is selected, modified, transfected, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression or replication, by the cell, of a gene, a polynucleotide such as a circular plasmid (e.g., pAVEC) or RNA or a protein. For example, a host cell may be a mammalian cell or bacterial cell (e.g., E. coli) or any isolated cell capable of maintaining pAVEC plasmid and, in an embodiment of the invention, promoting expression of a polypeptide encoded by a polynucleotide in the plasmid, e.g., an immunoglobulin chain. Examples of mammalian host cells include, by way of nonlimiting example, Chinese hamster ovary (CHO) cells, CHO-K1 cells, CHO-DXB-11 cells, CHO-DG44 cells, bovine mammary epithelial cells, mouse Sertoli cells, canine kidney cells, buffalo rat liver cells, human lung cells, mouse mammary tumor cells, rat fibroblasts, bovine kidney (MDBK) cells, NSO cells, SP2 cells, TRI cells, MRC 5 cells, FS4 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma (e.g., Hep G2) cells, A549 cells, etc. In one embodiment, the mammalian host cell is a human host cell. Mammalian host cells can be cultured according to methods known in the art (see, e.g., J. Immunol. Methods 56:221 (1983), Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. Oxford University Press, New York (1992)). Examples of suitable E. coli include DH1, DH5, DH5alpha, XL1-Blue, SURE, SCS110, OneShot Top 10, and HB101. In an embodiment of the invention an pAVEC plasmid of the present invention is maintained ectopically in a host cell and/or integrated into chromosomal DNA of the host cell. Such host cells and methods of use thereof, e.g., as discussed herein, form part of the present invention.

[0025] Vectors of the invention, such as pAVEC, may be introduced into host cells according to any of the many techniques known in the art, e.g., dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, electoporation, calcium phosphate co-precipitation, lipofection, direct microinjection of the vector into nuclei, or any other means appropriate for a given host cell type.

[0026] A “cassette” or an “expression cassette” refers to a DNA coding sequence or segment of DNA that codes for an expression product (e.g., peptide or RNA) that can be inserted into a vector, e.g., at defined restriction sites. The expression cassette may comprise a promoter and/or a terminator and/or polyA signal operably linked to the DNA coding sequence.

[0027] In general, a “promoter” or “promoter sequence” is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter sequence is, in general, bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. Within the promoter sequence may be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operably associated with or operably linked to other expression control sequences, including enhancer and repressor sequences or with a nucleic acid to be expressed. An expression control sequence is operably associated with or operably linked to a promoter if it regulates expression from said promoter.

[0028] Promoters which may be used to control gene expression include, but are not limited to, SRα promoter (Takebe et al., Molec. and Cell. Bio. 8:466-472 (1988)), the human CMV immediate early promoter (Boshart et al., Cell 41:521-530 (1985); Foecking et al., Gene 45:101-105 (1986)), the mouse CMV immediate early promoter, the SV40 early promoter region (Benoist et al., Nature 290:304-310 (1981)), the Orgyia pseudotsugata immediate early promoter, the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978)), or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)); and promoter elements from yeast or other fungi such as the GAL1, GAL4 or GAL10 promoter, the ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or the alkaline phosphatase promoter.

[0029] Viral long terminal repeat promoters such as the mouse mammary tumor virus long terminal repeat (MMTV-LTR) (Fasel et al., EMBO J. 1(1):3-7 (1982)), the moloney murine sarcoma virus long terminal repeat (Reddy et al., Proc. Natl. Acad. Sci. USA 77(9): 5234-5238 (1980)), the moloney murine leukemia virus long terminal repeat (Van Beveren et al., Proc. Natl. Acad. Sci. USA 77(6): 3307-3311 (1980)), the HIV LTR (Genbank Accession No. AB100245), the bovine foamy virus LTR (Genbank Accession No. NC_—001831), RSV 5′-LTR (Genbank Accession No. K00087), the HIV-2 LTR (Genbank Accession No. NC_—001722), an avian retroviral LTR (Ju et al., Cell 22: 379-386 (1980)) and the human herpesvirus LTR (Genbank Accession No. NC_—001806) may be included in the vectors of the present invention.

[0030] Other acceptable promoters include the human CMV5 promoter, the murine CMV promoter, the EF1α promoter, the SV40 promoter, a hybrid CMV promoter for liver specific expression (e.g., made by conjugating CMV immediate early promoter with the transcriptional promoter elements of either human α1-antitrypsin (HAT) or albumin (HAL) promoter), or promoters for hepatoma specific expression (e.g., wherein the transcriptional promoter elements of either human albumin (HAL; about 1000 bp) or human α1-antitrypsin (HAT, about 2000 bp) are combined with a 145 bp long enhancer element of human α1-microglobulin and bikunin precursor gene (AMBP); HAL-AMBP and HAT-AMBP).

[0031] In addition, bacterial promoters, such as the T7 RNA Polymerase promoter or the tac promoter, may be used to control expression.

[0032] In one embodiment, the promoter is the human CMV (hCMV) promoter. The hCMV promoter provides a high level of expression in a variety of mammalian cell types.

[0033] A coding sequence is “under the control of”, “functionally associated with”, “operably linked to” or “operably associated with” transcriptional and translational control sequences in a cell when the sequences direct or regulate expression of the sequence. For example, a promoter operably linked to a gene will direct RNA polymerase mediated transcription of the coding sequence into RNA, preferably mRNA, which may then be spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence. A terminator/polyA signal operably linked to a gene terminates transcription of the gene into RNA and directs addition of a polyA signal onto the RNA.

[0034] The terms “express” and “expression” mean allowing or causing the information in a gene, RNA or DNA sequence to become manifest; for example, producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene. “Express” and “expression” include transcription of DNA to RNA and of RNA to protein. A DNA sequence is expressed in or by a cell to form an “expression product” such as an RNA (e.g., mRNA) or a protein. The expression product itself may also be said to be “expressed” by the cell.

[0035] The term “transformation” means the introduction of a nucleic acid into a cell. The introduced gene or sequence may be called a “clone”. A host cell that receives the introduced DNA or RNA has been “transformed” and is a “transformant” or a “clone.” The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or from cells of a different genus or species. Examples of transformation methods which are very well known in the art include liposome delivery, electroporation, CaPO₄ transformation, DEAE-Dextran transformation, microinjection and viral infection.

[0036] The present invention includes vectors which comprise polynucleotides of the invention. The term “vector” may refer to a vehicle (e.g., a plasmid) by which a DNA or RNA sequence can be introduced into a host cell, so as to transform the host and, optionally, promote expression and/or replication of the introduced sequence.

[0037] The polynucleotides of the invention may be expressed in an expression system. The term “expression system” means a host cell and compatible vector which, under suitable conditions, can express a protein or nucleic acid which is carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors such as plasmids, cosmids, BACs, YACs and viruses such as adenovirus and adenovirus associated virus (AAV).

[0038] The invention provides the a plasmid which comprises the following multiple cloning site:

[0039] EcoRI, XhoI, PaeR7I, Bs120I, BstZ17I, Bst1107I, PstI, BamH1, e.g., comprising the relative spacing between these restriction sites as indicated in FIG. 1, e.g., wherein overlapping restriction sites with the multiple cloning site is characterized as follows:

[0040]

[0041] In an embodiment of the invention, the multiple cloning site comprises the nucleotide sequence:

[0042]

	(SEQ ID NO: 1)
	GAATTCTCTC GAGTGGGCCC AGTATACACT GCAGGGATCC

[0043] In the embodiment of the invention, the pAVEC plasmid of the present invention is characterized by the plasmid map set forth in FIG. 1.

[0044] In an embodiment of the invention, the plasmid is pAVEC which comprises the following nucleotide sequence:

[0045]

(SEQ ID NO: 2)
1	CTTGACTAGA GATCATAATC AGCCATACCA CATTTGTAGA
	GGTTTTACTT GCTTTAAAAA
61	ACCTCCCACA CCTCCCCCTG AACCTGAAAC ATAAAATGAA
	TGCAATTGTT GTTGTTAACT
121	TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG
	CATCACAAAT TTCACAAATA
181	AAGCATTTTT TTCACTGCAT TCTAGTTGTG GTTTGTCCAA
	ACTCATCAAT GTATCTTATC
241	ATGTCTGGAT CGATCCAAGC TTCGAGCACC GGTGGCCCGG
	GCCGGTCCGA CTAGTTACGA
301	TGTACGGGCC AGATATACGC GTTGACATTG ATTATTGACT
	AGTTATTAAT AGTAATCAAT
361	TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC
	GTTACATAAC TTACGGTAAA
421	TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG
	ACGTCAATAA TGACGTATGT
481	TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA
	TGGGTGGAGT ATTTACGGTA
541	AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA
	AGTACGCCCC CTATTGACGT
601	CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC
	ATGACCTTAT GGGACTTTCC
661	TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC
	ATGGTGATGC GGTTTTGGCA
721	GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA
	TTTCCAAGTC TCCACCCCAT
781	TGACGTCAAT GGGAGTTTGT TTTGGCACCA AAATCAACGG
	GACTTTCCAA AATGTCGTAA
841	CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA
	CGGTGGGAGG TCTATATAAG
901	CAGAGCTCTC TGGCTAACTA GAGAACCCAC TGCTTACTGG
	CTTATCGAAA TTAATACGAC
961	TCACTATAGC AATTGCACGT GTGGCCACAG GTAAGTTTAA
	AGCTCAGGTC GAGACCGGGC
1021	CTTTGTCCGG CGCTCCCTTG GAGCCTACCT AGACTCAGCC
	GGCTCTCCAC GCTTTGCCTG
1081	ACCCTGCTTG CTCAACTCTA CGTCTTTGTT TCGTTTTCTG
	TTCCTTTCTC TCCACAGGCT
1141	TAAGAATTCT CTCGAGTGGG CCCAGTATAC ACTGCAGGGA
	TCCAGATCCC CCTCGCTTTC
1201	TTGCTGTCCA ATTTCTATTA AAGGTTCCTT TGTTCCCTAA
	GTCCAACTAC TAAACTGGGG
1261	GATATTATGA AGGGCCTTGA GCATCTGGAT TCTGCCTAAT
	AAAAAACATT TATTTTCATT
1321	GCAATGATGT ATTTAAATTA TTTCTGAATA TTTTACTAAA
	AAGGGAATGT GGGAGGTCAG
1381	TGCATTTAAA ACATAAAGAA ATGAAGAGGG GGATCTGTCG
	ACAAGCTCTA GAGAGCTCCA
1441	TATGTGGCCA TCGCGATTAA TTAATCGCGA GCGGCCGCCA
	TATGTGGCCA ACGCGTGGTA
1501	CCGGCCGGCC GCGCGCTGTA CATCCGGATG ATCAGCTGAG
	GGTTTAAACG GCGCGCCTCT
1561	AGACAATTCT TGAAGACGAA AGGGCCTCGT GATACGCCTA
	TTTTTATAGG TTAATGTCAT
1621	GATAATAATG GTTTCTTAGA CGTCAGGTGG CACTTTTCGG
	GGAAATGTGC GCGGAACCCC
1681	TATTTGTTTA TTTTTCTAAA TACATTCAAA TATGTATCCG
	CTCATGAGAC AATAACCCTG
1741	ATAAATGCTT CAATAATATT GAAAAAGGAA GAGTATGAGT
	ATTCAACATT TCCGTGTCGC
1801	CCTTATTCCC TTTTTTGCGG CATTTTGCCT TCCTGTTTTT
	GCTCACCCAG AAACGCTGGT
1861	GAAAGTAAAA GATGCTGAAG ATCAGTTGGG TGCACGAGTG
	GGTTACATCG AACTGGATCT
1921	CAACAGCGGT AAGATCCTTG AGAGTTTTCG CCCCGAAGAA
	CGTTTTCCAA TGATGAGCAC
1981	TTTTAAAGTT CTGCTATGTG GCGCGGTATT ATCCCGTGTT
	GACGCCGGGC AAGAGCAACT
2041	CGGTCGCCGC ATACACTATT CTCAGAATGA CTTGGTTGAG
	TACTCACCAG TCACAGAAAA
2101	GCATCTTACG GATGGCATGA CAGTAAGAGA ATTATGCAGT
	GCTGCCATAA CCATGAGTGA
2161	TAACACTGCG GCCAACTTAC TTCTGACAAC GATCGGAGGA
	CCGAAGGAGC TAACCGCTTT
2221	TTTGCACAAC ATGGGGGATC ATGTAACTCG CCTTGATCGT
	TGGGAACCGG AGCTGAATGA
2281	AGCCATACCA AACGACGAGC GTGACACCAC GATGCCTGTA
	GCAATGGCAA CAACGTTGCG
2341	CAAACTATTA ACTGGCGAAC TACTTACTCT AGCTTCCCGG
	CAACAATTAA TAGACTGGAT
2401	GGAGGCGGAT AAAGTTGCAG GACCACTTCT GCGCTCGGCC
	CTTCCGGCTG GCTGGTTTAT
2461	TGCTGATAAA TCTGGAGCCG GTGAGCGTGG GTCTCGCGGT
	ATCATTGCAG CACTGGGGCC
2521	AGATGGTAAG CCCTCCCGTA TCGTAGTTAT CTACACGACG
	GGGAGTCAGG CAACTATGGA
2581	TGAACGAAAT AGACAGATCG CTGAGATAGG TGCCTCACTG
	ATTAAGCATT GGTAACTGTC
2641	AGACCAAGTT TACTCATATA TACTTTAGAT TGATTTAAAA
	CTTCATTTTT AATTTAAAAG
2701	GATCTAGGTG AAGATCCTTT TTGATAATCT CATGACCAAA
	ATCCCTTAAC GTGAGTTTTC
2761	GTTCCACTGA GCGTCAGACC CCGTAGAAAA GATCAAAGGA
	TCTTCTTGAG ATCCTTTTTT
2821	TCTGCGCGTA ATCTGCTGCT TGCAAACAAA AAAACCACCG
	CTACCAGCGG TGGTTTGTTT
2881	GCCGGATCAA GAGCTACCAA CTCTTTTTCC GAAGGTAACT
	GGCTTCAGCA GAGCGCAGAT
2941	ACCAAATACT GTCCTTCTAG TGTAGCCGTA GTTAGGCCAC
	CACTTCAAGA ACTCTGTAGC
3001	ACCGCCTACA TACCTCGCTC TGCTAATCCT GTTACCAGTG
	GCTGCTGCCA GTGGCGATAA
3061	GTCGTGTCTT ACCGGGTTGG ACTCAAGACG ATAGTTACCG
	GATAAGGCGC AGCGGTCGGG
3121	CTGAACGGGG GGTTCGTGCA CACAGCCCAG CTTGGAGCGA
	ACGACCTACA CCGAACTGAG
3181	ATACCTACAG CGTGAGCTAT GAGAAAGCGC CACGCTTCCC
	GAAGGGAGAA AGGCGGACAG
3241	GTATCCGGTA AGCGGCAGGG TCGGAACAGG AGAGCGCACG
	AGGGAGCTTC CAGGGGGAAA
3301	CGCCTGGTAT CTTTATAGTC CTGTCGGGTT TCGCCACCTC
	TGACTTGAGC GTCGATTTTT
3361	GTGATGCTCG TCAGGGGGGC GGAGCCTATG GAAAAACGCC
	AGCAACGCGG CCTTTTTACG
3421	GTTCCTGGCC TTTTGCTGGC CTTTTGCTCA CATGTTCTTT
	CCTGCGTTAT CCCCTGATTC
3481	TGTGGATAAC CGTATTACCG CCTTTGAGTG AGCTGATACC
	GCTCGCCGCA GCCGAACGAC
3541	CGAGCGCAGC GAGTCAGTGA GCGAGGAAGC GGAAGAGCGC
	TAGCAGCACG CCATAGTGAC
3601	TGGCGATGCT GTCGGAATGG ACGATATCCC GCAAGAGGCC
	CGGCAGTACC GGCATAACCA
3661	AGCCTATGCC TACAGCATCC AGGGTGACGG TGCCGAGGAT
	GACGATGAGC GCATTGTTAG
3721	ATTTCATACA CGGTGCCTGA CTGCGTTAGC AATTTAACTG
	TGATAAACTA CCGCATTAAA
3781	GCTCAGATCT GAGCTTGGGC AGAAATGGTT GAACTCCCGA
	GAGTGTCCTA CACCTAGGGG
3841	AGAAGCAGCC AAGGGGTTGT TTCCCACCAA GGACGACCCG
	TCTGCGCACA AACGGATGAG
3901	CCCATCAGAC AAAGACATAT TCATTCTCTG CTGCAAACTT
	GGCATAGCTC TGCTTTGCCT
3961	GGGGCTATTG GGGGAAGTTG CGGTTCGTGC TCGCAGGGCT
	CTCACCCTTG ACTCTTTTAA
4021	TAGCTCTTCT GTGCAAGATT ACAATCTAAA CAATTCGGAG
	AACTCGACCT TCCTCCTGAG
4081	GCAAGGACCA CAGCCAACTT CCTCTTACAA GCCGCATCGA
	TTTTGTCCTT CAGAAATAGA
4141	AATAAGAATG CTTGCTAAAA ATTATATTTT TACCAATAAG
	ACCAATCCAA TAGGTAGATT
4201	ATTAGTTACT ATGTTAAGAA ATGAATCATT ATCTTTTAGT
	ACTATTTTTA CTCAAATTCA
4261	GAAGTTAGAA ATGGGAATAG AAAATAGAAA GAGACGCTCA
	ACCTCAATTG AAGAACAGGT
4321	GCAAGGACTA TTGACCACAG GCCTAGAAGT AAAAAAGGGA
	AAAAAGAGTG TTTTTGTCAA
4381	AATAGGAGAC AGGTGGTGGC AACCAGGGAC TTATAGGGGA
	CCTTACATCT ACAGACCAAC
4441	AGATGCCCCC TTACCATATA CAGGAAGATA TGACTTAAAT
	TGGGATAGGT GGGTTACAGT
4501	CAATGGCTAT AAAGTGTTAT ATAGATCCCT CCCTTTTCGT
	GAAAGACTCG CCAGAGCTAG
4561	ACCTCCTTGG TGTATGTTGT CTCAAGAAGA AAAAGACGAC
	ATGAAACAAC AGGTACATGA
4621	TTATATTTAT CTAGGAACAG GAATGCACTT TTGGGGAAAG
	ATTTTCCATA CCAAGGAGGG
4681	GACAGTGGCT GGACTAATAG AACATTATTC TGCAAAAACT
	CATGGCATGA GTTATTATGA
4741	ATAGCCTTTA TTGGCCCAAC CTTGCGGTTC CCAGGGCTTA
	AGTAAGTTTT TGGTTACAAA
4801	CTGTTCTTAA AACGAGGATG TGAGACAAGT GGTTTCCTGA
	CTTGGTTTGG TATCAAAGGT
4861	TCTGATCTGA GCTCTGAGTG TTCTATTTTC CTATGTTCTT
	TTGGAATTTA TCCAAATCTT
4921	ATGTAAATGC TTATGTAAAC CAAGATATAA AAGAGTGCTG
	ATTTTTTGAG TAAACTTGCA
4981	ACAGTCCTAA CATTCACCTC TTGTGTGTTT GTGTCTGTTC
	GCCATCCCGT CTCCGCTCGT
5041	CACTTATCCT TCACTTTCCA GAGGGTCCCC CCGCAGACCC
	CGGCGACCCT CAGGTCGGCC
5101	GACTGCGGCA GCTGGCGCCC GAACAGGGAC CCTCGGATAA
	GTGACCCTTG TCTCTATTTC
5161	TACTATTTGG TGTTTGTCTT GTATTGTCTC TTTCTTGTCT
	GGCTATCATC ACAAGAGCGG
5221	AACGGACTCA CCATAGGGAC CAAGCTCAGA TCTGAGCTTG
	GGGGGGGGGA CAGCTCAGGG
5281	CTGCGATTTC GCGCCAAACT TGACGGCAAT CCTAGCGTGA
	AGGCTGGTAG GATTTTATCC
5341	CCGCTGCCAT CATGGTTCGA CCATTGAACT GCATCGTCGC
	CGTGTCCCAA AATATGGGGA
5401	TTGGCAAGAA CGGAGACCTA CCCTGGCCTC CGCTCAGGAA
	CGAGTTCAAG TACTTCCAAA
5461	GAATGACCAC AACCTCTTCA GTGGAAGGTA AACAGAATCT
	GGTGATTATG GGTAGGAAAA
5521	CCTGGTTCTC CATTCCTGAG AAGAATCGAC CTTTAAAGGA
	CAGAATTAAT ATAGTTCTCA
5581	GTAGAGAACT CAAAGAACCA CCACGAGGAG CTCATTTTCT
	TGCCAAAAGT TTGGATGATG
5641	CCTTAAGACT TATTGAACAA CCGGAATTGG CAAGTAAAGT
	AGACATGGTT TGGATAGTCG
5701	GAGGCAGTTC TGTTTACCAG GAAGCCATGA ATCAACCAGG
	CCACCTCAGA CTCTTTGTGA
5761	CAAGGATCAT GCAGGAATTT GAAAGTGACA CGTTTTTCCC
	AGAAATTGAT TTGGGGAAAT
5821	ATAAACTTCT CCCAGAATAC CCAGGCGTCC TCTCTGAGGT
	CCAGGAGGAA AAAGGCATCA
5881	AGTATAAGTT TGAAGTCTAC GAGAAGAAAG ACTAACAGGA
	AGATGCTTTC AAGTTCTCTG
5941	CTCCCCTCCT AAAGCTATGC ATTTTTATAA GACCATGGGA
	CTTTTGCTGG CTTTAGATCG
6001	ATCTTTGTGA AGGAACCTTA CTTCTGTGGT GTGACATAAT
	TGGACAAACT ACCTACAGAG
6061	ATTTAAAGCT CTAAGGTAAA TATAAAATTT TTAAGTGTAT
	AATGTGTTAA ACTACTGATT
6121	CTAATTGTTT GTGTATTTTA GATTCCAACC TATGGAACTG
	ATGAATGGGA GCAGTGGTGG
6181	AATGCCTTTA ATGAGGAAAA CCTGTTTTGC TCAGAAGAAA
	TGCCATCTAG TGATGATGAG
6241	GCTACTGCTG ACTCTCAACA TTCTACTCCT CCAAAAAAGA
	AGAGAAAGGT AGAAGACCCC
6301	AAGGACTTTC CTTCAGAATT GCTAAGTTTT TTGAGTCATG
	CTGTGTTTAG TAATAGAACT
6361	CTTGCTTGCT TTGCTATTTA CACCACAAAG GAAAAAGCTG
	CACTGCTATA CAAGAAAATT
6421	ATGGAAAAAT ATTCTGTAAC CTTTATAAGT AGGCATAACA
	GTTATAATCA TAACATACTG
6481	TTTTTTCTTA CTCCACACAG GCATAGAGTG TCTGCTATTA
	ATAACTATGC TCAAAAATTG
6541	TGTACCTTTA GCTTTTTAAT TTGTAAAGGG GTTAATAAGG
	AATATTTGAT GTATAGTGC

[0046] Any of several genes may be inserted into the plasmids of the present invention, for example, immunoglobulins. Plasmids of the present invention encoding any of the following target immunoglobulin amino acid sequences form part of the present invention. The present invention encompasses the product of any method wherein a polynucleotide or gene (e.g., encoding an immunoglobulin) is inserted into pAVEC and which, in the process, results in a loss of some pAVEC polynucleotide sequence, e.g., due to excision of a portion of the multiple cloning site so as to generate restriction enzymatically cleaved sites that are compatible for ligation of the introduced gene into the vector. The product of such a method may be referred to herein as a pAVEC vector that comprises a particular gene or polynucleotide.

[0047] The scope of the present invention includes a method for introducing a polynucleotide into the pAVEC plasmid comprising cleaving the plasmid, e.g., with one or more restriction endonucleases, e.g., in the plasmid multiple cloning site, and ligating the ends of the cleaved plasmid to the compatible ends of the introduced polynucleotide, e.g., with a DNA ligase to produce a closed recombined plasmid. Any recombined plasmid that is the product of such a method is part of the present invention. Compatible ends of polynucleotides are ends that can be joined together by a DNA ligase. In an embodiment of the invention, the ends are blunt or sticky ends, e.g., the result of cleavage by a restriction endonuclease such as EcoR1 or BamH1.

[0048] In an embodiment of the invention, the immunoglobulin is the mature or unprocessed version of any of the following polypeptides, e.g., the variable region only or the variable domain and the constant domain:

[0049]

[0050] See international application publication no. WO2003/100008 which is incorporated herein by reference in its entirety.

[0051] Embodiments of the invention include those wherein the plasmid includes more than one immunoglobulin, for example, a combination of any of those set forth herein (e.g., heavy chain Ig. #1.0 and light chain Ig. #1.0, or LCC and HCA; or LCF and HCA; or LCC and HCB).

[0052] In an embodiment of the invention the pAVEC plasmid contains a light chain immunoglobulin comprising the amino acid sequence:

[0053]

(SEQ ID NO: 11)
1	DIVMTQSPLS LPVTPGEPAS ISCRSSQSIV HSNGNTYLQW
	YLQKPGQSPQ
51	LLIYKVSNRL YGVPDRFSGS GSGTDFTLKI SRVEAEDVGV
	YYCFQGSHVP
101	WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL
	LNNFYPREAK
151	VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD
	YEKHKVYACE
201	VTHQGLSSPV TKSFNRGEC;

and/or, a heavy chain immunoglobulin comprising the amino acid sequence:

[0054]

(SEQ ID NO: 12);
1	QVQLQESGPG LVKPSETLSL TCTVSGYSIT GGYLWNWIRQ
	PPGKGLEWIG
51	YISYDGTNNY KPSLKDRVTI SRDTSKNQFS LKLSSVTAAD
	TAVYYCARYG
101	RVFFDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA
	ALGCLVKDYF
151	PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS
	SSLGTQTYIC
201	NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP APELLGGPSV
	FLFPPKPKDT
251	LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK
	PREEQYNSTY
301	RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK
	GQPREPQVYT
351	LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN
	YKTTPPVLDS
401	DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS
	LSLSPGK;

[0055] or a variable domain thereof or a light chain and/or heavy chain immunoglobulin comprising one or more CDRs (e.g., 3) of the light and/or heavy chain, e.g., those which are underscored in the sequences above.

[0056] In an embodiment of the invention, the pAVEC vector comprises polynucleotides encoding the light and/or heavy immunoglobulin chains of antibodies such as Abciximab, Adalimumab, Alemtuzumab, Basiliximab, Bevacizumab, Cetuximab, Certolizumab pegol, Dalotuzumab, Daclizumab, Denosumab, Eculizumab, Efalizumab, Gemtuzumab, Ibritumomab tiuxetan, Infliximab, Muromonab-CD3, Natalizumab, Omalizumab, Palivizumab, Panitumumab, Ranibizumab, Rituximab, Robatumumab, Tositumomab, ALD518 or Trastuzumab. The sequences of such antibodies are known in the art. In an embodiment of the invention, the pAVEC vector comprises a heavy and/or light chain immunoglobulin of an antibody or antigen-binding fragment thereof that binds specifically to an antigen (e.g., from any mammal such as a human or canine or monkey or rat or rabbit or mouse); e.g., an antigen such as VEGF, VEGFR, EGF, EGFR, PD-1, TNFalpha, TGFbeta, TRAIL-R1, TSLP, Nav1.7, Nav1.8, ERK, MEK, TRAIL-R2, IL-10, IL-6, IL-6R, IGF1R, IL-23p19, IL23R, PCSK9, CD20, RANKL, RANK, CD33, CD11a, ErbB2, IgE, a G-protein coupled receptor (GPCR) an HIV antigen, an HCV antigen or a respiratory syncytial virus (RSV) antigen; or an antigen-binding fragment thereof, or a variable domain of any of such antibodies or antigen-binding fragments or a light chain and/or heavy chain immunoglobulin comprising one or more CDRs (e.g., 3) of the light and/or heavy chain of any of such antibodies or antigen-binding fragments. In an embodiment of the invention, the species from which the antigen is derived is the same or different from that of the light and/or heavy chain constant domain; and/or immunoglobulin frameworks.

[0057] In an embodiment of the invention, the pAVEC vector comprises a light and/or heavy chain immunoglobulin comprising a member selected from the group consisting of:

[0058]

(a) an immunoglobulin heavy chain comprising the amino acid sequence:
(SEQ ID NO: 13)
E V Q L Q Q S G A E L V K P G A S V T L S C T A S G F N I K D T Y
M H W V N Q R P E Q G L V W I G R I D P A N G H T E Y D P K F Q D
K A T I T T D T S S N T A Y L H L S S L T S G D T A V Y Y C A R S
Y F G S I F A Y W G Q G T L V T V S A;
(b) an immunoglobulin light chain comprising the amino acid sequence:
(SEQ ID NO: 14)
Q I V L T Q S P A I M S A S P G E K V T I S C S A S S S V S Y L Y
W Y Q Q K P G S S P K P W I F R S S H R A S G V P A R F S G S G S
G T S Y S L T I S S M E A E D A A T Y Y C H Q Y Q S Y P P T F G G
G T K L E I K R A;
(c) an immunoglobulin heavy chain comprising the amino acid sequence:
(SEQ ID NO: 15)
E V Q L Q Q S G A D L V K P G A S V K L S C T A S G F N I K D T Y
I H W V K Q R P E Q G L E W I G R I D P A N G H T E Y D P K F Q G
R A T L T T D T S S N T A Y L Q L F S L T S E D S A V Y F C A R S
Y Y G S I F A Y W G Q G T L V T V S A;
(d) an immunoglobulin light chain comprising the amino acid sequence:
(SEQ ID NO: 16)
Q I V L T Q S P A I M S A S P G E K V T I S C S A S S S V S Y L F
W Y Q Q K P G S S P K P W I F R T S Y L A S G V P A R F S G S G S
G T S F S L T I S S M E A E D A A T Y Y C H Q Y H T Y P P T F G G
G T K L E I K R A;
(e) an immunoglobulin heavy chain comprising the amino acid sequence:
(SEQ ID NO: 17)
E V Q L Q Q S G A E L V R S G A S V K L S C T T S G F N I K D Y Y
I H W V K Q R P E Q G L E W I G W I D P E N G D T E Y A P K F Q G
K A T M T A D T S S N T A Y L Q L S S L T S A D T A V Y Y C N A
Y Y R Y D D G T W F P Y W G Q G T L V T V S A;
(f) an immunoglobulin light chain comprising the amino acid sequence:
(SEQ ID NO: 18)
D I Q L T Q S P A S L S A S V G E T V T I T C R A S G N I H S Y L
A W Y Q Q K Q G K S P Q F L V D N A K T L P D G V P S R F S V S G
S G T Q Y S L K I N S L Q P E D F G T Y Y C Q H F W N T P W T F G
G G T K L E I K R A;
(g) an immunoglobulin heavy chain comprising the amino acid sequence:
(SEQ ID NO: 19)
E V L L Q Q S V A E L V R P G A S V R L S C T A S G F N I K D T Y
I H W V R Q R P E Q G L E W F G W I D P A N G Y T K Y A P N F Q G
K A T L T T D T S S N T A Y L H L S S L T S E D S A I Y Y C A R G
Y Y R Y Y S L D Y W G Q G T S V T V S S;
(h) an immunoglobulin light chain comprising the amino acid sequence:
(SEQ ID NO: 20)
D I Q M T Q T T S S L S A S L G D R V T I S C R A S Q D I S N Y L
N W Y Q Q K P D G T V K L L I Y Y S S R L H S G V P S R F S G R G
S G T D Y S L T I S T L E Q E D I A T Y F C Q Q G K T L P L T F G
A G T K L E L K R A;
(i) an immunoglobulin heavy chain comprising the amino acid sequence:
(SEQ ID NO: 21)
E V Q L V D S G G G L V Q P G R S L K L S C A A S G F T F S N H D
M A W V R Q A P T K G L E W V A S I T P S G G T T Y Y R D S V E G
R F T V S R D N V K S S L H L Q M D S L T S E D T A T Y Y C A R Q
N Y Y D G S Y Y Y G L Y Y F D Y W G Q G V M V T V S S;
(j) an immunoglobulin light chain comprising the amino acid sequence:
(SEQ ID NO: 22)
D V L M T Q T P V S L P V S L G G Q V S I S C R S S Q S L V Y S D
G N T Y L H W Y L Q K P G Q S P Q L L I Y R V S N R F S G V P D R
F S G S G S G T D F T L K I S R V E P E D L G L Y Y C L Q S T H F
P P T F G S G T K L E I K R A;
(k) an immunoglobulin heavy chain comprising the amino acid sequence:
(SEQ ID NO: 23)
E V Q L Q Q S G P E L V K P G A S V K I S C K V S G Y T F T D Y Y
M N W V K Q S H G K S L E W I G D I N P N N G G A I Y N Q K F K G
K A T L T V D K S S S I A Y M E L R S L T S E D S A V Y Y C T S G
I I T E I A E D F W G Q G T T L T V S S;
and
(l) an immunoglobulin light chain comprising the amino acid sequence:
(SEQ ID NO: 24)
D I V M T Q S Q K F M S T S V G D R V S V T C K A S Q N V G T N V
V W Y Q Q K P G Q S P K A L I H S A S Y R Y S G V P D R F K G S G
S G T D F T L T I T N V Q S E D L A G F F C Q Q Y K T Y P Y T F G
G G T Q L E I K R A;
or an immunoglobulin comprising one or more CDRs (e.g., 3) of the
light and/or heavy chains set forth above.

[0059] A further aspect of the present invention relates to a method for the production of a recombinant protein and/or propagation of a particular polynucleotide using a pAVEC plasmid. For example, in an embodiment of the invention, a method for expressing a protein comprises culturing a host cell (e.g., a CHO cell) comprising a pAVEC plasmid having a polynucleotide that encodes a polypeptide (e.g., that is operably linked to a promoter such as the hCMV promoter of pAVEC) under appropriate conditions to enable growth of the host cell comprising the plasmid and expression of the recombinant protein, e.g., a method that comprises the steps of:

[0060] a) introducing a polynucleotide encoding a polypeptide of interest (e.g., an immunoglobulin chain) into a pAVEC plasmid (e.g., SEQ ID NO:1), e.g., that is operably linked to a promoter such as the hCMV promoter of pAVEC;

[0061] As was discussed above, introducing the polynucleotide into the pAVEC plasmid may result in loss of some pAVEC polynucleotide sequence, e.g., due to excision of a portion of the multiple cloning site so as to generate restriction enzymatically cleaved sites that are compatible for ligation to the ends of the introduced polynucleotide into the plasmid.

[0062] b) transfecting a host cell with a pAVEC plasmid comprising the polynucleotide encoding the protein of interest;

[0063] The plasmid may be propagated in the host cells either ectopically as an autonomously replicating element (e.g., wherein the plasmid has a high copy number, e.g., about 2, 3, 4, 5, 10, 20 or 50) or integrated into a host cell chromosome.

[0064] c) culturing the cell under appropriate conditions to enable growth of the host cell comprising the plasmid and expression of the recombinant protein; and, optionally

[0065] d) harvesting the polypeptide produced.

[0066] Methods for harvesting (isolating and/or purifying) a given protein from, e.g., a cell, a cell culture or the medium in which cells have been cultured are well known in the art. By way of nonlimiting example, proteins can be isolated and/or purified from biological material by salt or alcohol precipitation (e.g., ammonium sulfate precipitation or ethanol precipitation), affinity chromatography (e.g., used in conjunction with a purification tag); fractionation on immunoaffinity or ion-exchange columns; high pressure liquid chromatography (HPLC); reversed-phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing;); isoelectric focusing; countercurrent distribution; SDS-PAGE; gel filtration (using, e.g., Sephadex G-75); and protein A Sepharose columns to remove contaminants such as IgG. Such purification methods are well known in the art and are disclosed, e.g., in “Guide to Protein Purification”, Methods in Enzymology, Vol. 182, M. Deutscher, Ed., 1990, Academic Press, New York, N.Y.

[0067] Growth of mammalian cells in liquid aqueous culture is well known in the art. Examples of mammalian cell culture growth media which are known in the art include EX-CELL® ACF growth medium, CHO medium (Sigma-Aldrich (St. Louis, Mo.); discussed further below), DMEM, DMEM/F-12, F-10 Nutrient Mixture, RPMI Medium 1640, F-12 Nutrient Mixture, Medium 199, Eagle's MEM, RPMI, 293 media, and Iscove's Media.

[0068] Cell growth can be performed in any of several systems. For example, cell growth can be done in a simple flask, e.g., a glass shake flask. Other systems include tank bioreactors, bag bioreactors and disposable bioreactors. A tank bioreactor includes, typically, a metal vessel (e.g., a stainless steel jacketed vessel) in which cells are growth in a liquid medium. Tank bioreactors can be used for a wide range of culture volumes (e.g., 100 l, 150 l, 10000 l, 15000 l). Tank bioreactors often have additional features for controlling cell growth conditions, including means for temperature control, medium agitation, controlling sparge gas concentrations, controlling pH, controlling O₂ concentration, removing samples from the medium, reactor weight indication and control, cleaning hardware, sterilizing the hardware, piping or tubing to deliver all services, adding media, control pH, control solutions, and control gases, pumping sterile fluids into the growth vessel and, supervisory control and a data acquisition. Classifications of tank bioreactor include stirred tank reactors wherein mechanical stirrers (e.g., impellers) are used to mix the reactor to distribute heat and materials (such as oxygen and substrates). Bubble column reactors are tall reactors which use air alone to mix the contents. Air lift reactors are similar to bubble column reactors, but differ by the fact that they contain a draft tube. The draft tube is typically an inner tube which improves circulation and oxygen transfer and equalizes shear forces in the reactor. In fluidized bed reactors, cells are “immobilized” on small particles which move with the fluid. The small particles create a large surface area for cells to stick to and enable a high rate of transfer of oxygen and nutrients to the cells. In packed bed reactors cells are immobilized on large particles. These particles do not move with the liquid. Packed bed reactors are simple to construct and operate but can suffer from blockages and from poor oxygen transfer. A disposable bioreactor is a disposable, one-time use bioreactor. Often, disposable bioreactors possess features similar to non-disposable bioreactors (e.g., agitation system, sparge, probes, ports, etc.).

[0069] Particularly where a polypeptide is isolated from a cellular or tissue source, it is preferable to include one or more inhibitors of proteolytic enzymes in the assay system, such as phenylmethanesulfonyl fluoride (PMSF), PEFABLOC® SC protease inhibitor, pepstatin, leupeptin, chymostatin and EDTA.

[0070] In some embodiments, the protein of interest is with a second polypeptide or polynucleotide moiety, which may be referred to as a “tag” or “marker”. A tag may be used, for example, to facilitate purification or detection of the polypeptide after expression. A fused polypeptide may be constructed, for example, by in-frame insertion of a polynucleotide encoding the tag on the 5′ or 3′ end of the polynucleotide encoding the polypeptide to be expressed. The fused polynucleotide may then be expressed in the expression system of the invention. Such tags include glutathione-S-transferase (GST), hexahistidine (His6) tags, maltose binding protein (MBP) tags, haemagglutinin (HA) tags, cellulose binding protein (CBP) tags and myc tags. Detectable tags such as ³²P, ³⁵S, ³H, ^99mTc, ¹²³I, ¹¹¹In, ⁶⁸Ga, ¹⁸F, ¹²⁵I, ¹³¹I, ^113mIn, ⁷⁶Br, ⁶⁷Ga, ^99mTc, ¹²³I, ¹¹¹In and ⁶⁸Ga may also be used to label the polypeptides and polynucleotides of the invention. Methods for constructing and using such fusions are very conventional and well known in the art.

[0071] One skilled in the art appreciates that purification methods suitable for the polypeptide of interest may require modification to account for changes in the character of the polypeptide upon expression in recombinant cell culture.

[0072] The present invention also relates to a method for the introducing a polynucleotide into a pAVEC plasmid comprising transforming a host cell with the plasmid, e.g., comprising the steps of:

[0073] a) cleaving the pAVEC plasmid at the site into which insertion of the polynucleotide is desired (e.g., using a restriction enzyme, e.g., that generates ends that are compatible for hybridization with and subsequent ligation ends of the polynucleotide to be inserted). The ends of the polynucleotide to be inserted may also be generated by a process including cleavage with a restriction enzyme that generates ends compatible with the ends of the cleaved plasmid.

[0074] As was discussed above, introducing the polynucleotide into the pAVEC plasmid may result in loss of some pAVEC polynucleotide sequence, e.g., due to excision of a portion of the multiple cloning site so as to generate restriction enzymatically cleaved sites that are compatible for ligation to the ends of the introduced polynucleotide into the plasmid.

[0075] b) ligating ends of the cleaved pAVEC with the ends of the polynucleotide (e.g., using a DNA ligase) to generate a final circular recombined plasmid;

[0076] b) the recombined plasmid may then be transformed into a host cell wherein the plasmid and the added polynucleotide is propagated. The plasmid may be propagated in the host cells either ectopically as an autonomously replicating element (e.g., wherein the plasmid has a high copy number, e.g., about 2, 3, 4, 5, 10, 20 or 50) or integrated into a host cell chromosome.

[0077] The pAVEC plasmid vectors of the invention may be provided in a kit. The kits of the invention may include, in addition to the plasmid vector, any reagent which may be employed in the use of the plasmid vector. In one embodiment, the kit includes reagents necessary for transformation of the vectors into bacterial and/or mammalian host cells. For example, the kit may include reagents for a calcium phosphate transformation procedure: calcium chloride, buffer (e.g., 2X HEPES buffered saline), and sterile, distilled water. In another embodiment, the kit includes reagents for a DEAE-Dextran transformation: Chloroquine in PBS, DEAE-dextran in PBS and Phosphate buffered saline. In yet another embodiment, reagents for a liposome transformation are included in the kit: Liposomes extruded from DOTAP/cholesterol extruded liposomes. For example, the kit may include the cationic lipid-based transfection reagent LIPOFECTAMINE™ transfection reagent (Invitrogen Life Technologies; Carlsbad, Calif.).

[0078] The kit may include reagents required for bacterial transformation of the vectors of the invention. For example, the kit may include transformation competent bacteria (e.g., DH1, DH5, DH5α, XL1-Blue, SURE, SCS110, OneShot Top 10, or HB101).

[0079] The kit may include growth media or reagents required for making growth media. For example, in one embodiment, the kit can include fetal calf serum or DMEM (Dulbecco/Vogt modified Eagle's (Harry Eagle) minimal essential medium) for growth of mammalian cells. In another embodiment, the kit can contain powdered Luria broth media or Luria broth plates containing an appropriate antibiotic (e.g., ampicillin or kanamycin) for growing bacteria.

[0080] Components supplied in the kit may be provided in appropriate vials or containers (e.g., plastic or glass vials). The kit can include appropriate label directions for storage, and appropriate instructions for usage.

[0081] The following example is provided to further describe the present invention and should not be construed as a limitation thereof. The scope of the present invention includes any and all of the methods which are set forth below in the following example.

[0082] The sequence of the multiple cloning site (MCS):

[0083]

[0084]

	(SEQ ID NO: 1)
	GAATTCTCTC GAGTGGGCCC AGTATACACT GCAGGGATCC;

was synthesized by polymerase chain reaction (PCR). The PCR product was later digested by the restriction enzymes, EcoRI and BamHI. The vector, pAIL17L (FIG. 2) was also digested by the restriction enzymes EcoRI and BamHI. Finally the digested PCR product was ligated to the digested vector and the transformants were screened for the vector, pAVEC. The multiple cloning site in pAVEC was later analyzed by sequencing for the integrity of the site.

[0085]

Sequence of pAIL17L:
(SEQ ID NO: 25)
1	GCACTA TACATC AAATAT TCCTTA TTAACC CCTTTA CAAATT AAAAAG CTAAAG GTACAC
61	AATTTT TGAGCA TAGTTA TTAATA GCAGAC ACTCTA TGCCTG TGTGGA GTAAGA AAAAAC
121	AGTATG TTATGA TTATAA CTGTTA TGCCTA CTTATA AAGGTT ACAGAA TATTTT TCCATA
181	ATTTTC TTGTAT AGCAGT GCAGCT TTTTCC TTTGTG GTGTAA ATAGCA AAGCAA GCAAGA
241	GTTCTA TTACTA AACACA GCATGA CTCAAA AAACTT AGCAAT TCTGAA GGAAAG TCCTTG
301	GGGTCT TCTACC TTTCTC TTCTTT TTTGGA GGAGTA GAATGT TGAGAG TCAGCA GTAGCC
361	TCATCA TCACTA GATGGC ATTTCT TCTGAG CAAAAC AGGTTT TCCTCA TTAAAG GCATTC
421	CACCAC TGCTCC CATTCA TCAGTT CCATAG GTTGGA ATCTAA AATACA CAAACA ATTAGA
481	ATCAGT AGTTTA ACACAT TATACA CTTAAA AATTTT ATATTT ACCTTA GAGCTT TAAATC
541	TCTGTA GGTAGT TTGTCC AATTAT GTCACA CCACAG AAGTAA GGTTCC TTCACA AAGATC
601	GATCTA AAGCCA GCAAAA GTCCCA TGGTCT TATAAA AATGCA TAGCTT TAGGAG GGGAGC
661	AGAGAA CTTGAA AGCATC TTCCTG TTAGTC TTTCTT CTCGTA GACTTC AAACTT ATACTT
721	GATGCC TTTTTC CTCCTG GACCTC AGAGAG GACGCC TGGGTA TTCTGG GAGAAG TTTATA
781	TTTCCC CAAATC AATTTC TGGGAA AAACGT GTCACT TTCAAA TTCCTG CATGAT CCTTGT
841	CACAAA GAGTCT GAGGTG GCCTGG TTGATT CATGGC TTCCTG GTAAAC AGAACT GCCTCC
901	GACTAT CCAAAC CATGTC TACTTT ACTTGC CAATTC CGGTTG TTCAAT AAGTCT TAAGGC
961	ATCATC CAAACT TTTGGC AAGAAA ATGAGC TCCTCG TGGTGG TTCTTT GAGTTC TCTACT
1021	GAGAAC TATATT AATTCT GTCCTT TAAAGG TCGATT CTTCTC AGGAAT GGAGAA CCAGGT
1081	TTTCCT ACCCAT AATCAC CAGATT CTGTTT ACCTTC CACTGA AGAGGT TGTGGT CATTCT
1141	TTGGAA GTACTT GAACTC GTTCCT GAGCGG AGGCCA GGGTAG GTCTCC GTTCTT GCCAAT
1201	CCCCAT ATTTTG GGACAC GGCGAC GATGCA GTTCAA TGGTCG AACCAT GATGGC AGCGGG
1261	GATAAA ATCCTA CCAGCC TTCACG CTAGGA TTGCCG TCAAGT TTGGCG CGAAAT CGCAGC
1321	CCTGAG CTGTCC CCCCCC CCAAGC TCAGAT CTGAGC TTGGTC CCTATG GTGAGT CCGTTC
1381	CGCTCT TGTGAT GATAGC CAGACA AGAAAG AGACAA TACAAG ACAAAC ACCAAA TAGTAG
1441	AAATAG AGACAA GGGTCA CTTATC CGAGGG TCCCTG TTCGGG CGCCAG CTGCCG CAGTCG
1501	GCCGAC CTGAGG GTCGCC GGGGTC TGCGGG GGGACC CTCTGG AAAGTG AAGGAT AAGTGA
1561	CGAGCG GAGACG GGATGG CGAACA GACACA AACACA CAAGAG GTGAAT GTTAGG ACTGTT
1621	GCAAGT TTACTC AAAAAA TCAGCA CTCTTT TATATC TTGGTT TACATA AGCATT TACATA
1681	AGATTT GGATAA ATTCCA AAAGAA CATAGG AAAATA GAACAC TCAGAG CTCAGA TCAGAA
1741	CCTTTG ATACCA AACCAA GTCAGG AAACCA CTTGTC TCACAT CCTCGT TTTAAG AACAGT
1801	TTGTAA CCAAAA ACTTAC TTAAGC CCTGGG AACCGC AAGGTT GGGCCA ATAAAG GCTATT
1861	CATAAT AACTCA TGCCAT GAGTTT TTGCAG AATAAT GTTCTA TTAGTC CAGCCA CTGTCC
1921	CCTCCT TGGTAT GGAAAA TCTTTC CCCAAA AGTGCA TTCCTG TTCCTA GATAAA TATAAT
1981	CATGTA CCTGTT GTTTCA TGTCGT CTTTTT CTTCTT GAGACA ACATAC ACCAAG GAGGTC
2041	TAGCTC TGGCGA GTCTTT CACGAA AAGGGA GGGATC TATATA ACACTT TATAGC CATTGA
2101	CTGTAA CCCACC TATCCC AATTTA AGTCAT ATCTTC CTGTAT ATGGTA AGGGGG CATCTG
2161	TTGGTC TGTAGA TGTAAG GTCCCC TATAAG TCCCTG GTTGCC ACCACC TGTCTC CTATTT
2221	TGACAA AAACAC TCTTTT TTCCCT TTTTTA CTTCTA GGCCTG TGGTCA ATAGTC CTTGCA
2281	CCTGTT CTTCAA TTGAGG TTGAGC GTCTCT TTCTAT TTTCTA TTCCCA TTTCTA ACTTCT
2341	GAATTT GAGTAA AAATAG TACTAA AAGATA ATGATT CATTTC TTAACA TAGTAA CTAATA
2401	ATCTAC CTATTG GATTGG TCTTAT TGGTAA AAATAT AATTTT TAGCAA GCATTC TTATTT
2461	CTATTT CTGAAG GACAAA ATCGAT GCGGCT TGTAAG AGGAAG TTGGCT GTGGTC CTTGCC
2521	TCAGGA GGAAGG TCGAGT TCTCCG AATTGT TTAGAT TGTAAT CTTGCA CAGAAG AGCTAT
2581	TAAAAG AGTCAA GGGTGA GAGCCC TGCGAG CACGAA CCGCAA CTTCCC CCAATA GCCCCA
2641	GGCAAA GCAGAG CTATGC CAAGTT TGCAGC AGAGAA TGAATA TGTCTT TGTCTG ATGGGC
2701	TCATCC GTTTGT GCGCAG ACGGGT CGTCCT TGGTGG GAAACA ACCCCT TGGCTG CTTCTC
2761	CCCTAG GTGTAG GACACT CTCGGG AGTTCA ACCATT TCTGCC CAAGCT CAGATC TGAGCT
2821	TTAATG CGGTAG TTTATC ACAGTT AAATTG CTAACG CAGTCA GGCACC GTGTAT GAAATC
2881	TAACAA TGCGCT CATCGT CATCCT CGGCAC CGTCAC CCTGGA TGCTGT AGGCAT AGGCTT
2941	GGTTAT GCCGGT ACTGCC GGGCCT CTTGCG GGATAT CGTCCA TTCCGA CAGCAT CGCCAG
3001	TCACTA TGGCGT GCTGCT AGCGCT CTTCCG CTTCCT CGCTCA CTGACT CGCTGC GCTCGG
3061	TCGTTC GGCTGC GGCGAG CGGTAT CAGCTC ACTCAA AGGCGG TAATAC GGTTAT CCACAG
3121	AATCAG GGGATA ACGCAG GAAAGA ACATGT GAGCAA AAGGCC AGCAAA AGGCCA GGAACC
3181	GTAAAA AGGCCG CGTTGC TGGCGT TTTTCC ATAGGC TCCGCC CCCCTG ACGAGC ATCACA
3241	AAAATC GACGCT CAAGTC AGAGGT GGCGAA ACCCGA CAGGAC TATAAA GATACC AGGCGT
3301	TTCCCC CTGGAA GCTCCC TCGTGC GCTCTC CTGTTC CGACCC TGCCGC TTACCG GATACC
3361	TGTCCG CCTTTC TCCCTT CGGGAA GCGTGG CGCTTT CTCATA GCTCAC GCTGTA GGTATC
3421	TCAGTT CGGTGT AGGTCG TTCGCT CCAAGC TGGGCT GTGTGC ACGAGC CCCCCG TTCAGC
3481	CCGACC GCTGCG CCTTAT CCGGTA ACTATC GTCTTG AGTCCA ACCCGG TAAGAC ACGACT
3541	TATCGC CACTGG CAGCAG CCACTG GTAACA GGATTA GCAGAG CGAGGT ATGTAG GCGGTG
3601	CTACAG AGTTCT TGAAGT GGTGGC CTAACT ACGGCT ACACTA GAAGGA CAGTAT TTGGTA
3661	TCTGCG CTCTGC TGAAGC CAGTTA CCTTCG GAAAAA GAGTTG GTAGCT CTTGAT CCGGCA
3721	AACAAA CCACCG CTGGTA GCGGTG GTTTTT TTGTTT GCAAGC AGCAGA TTACGC GCAGAA
3781	AAAAAG GATCTC AAGAAG ATCCTT TGATCT TTTCTA CGGGGT CTGACG CTCAGT GGAACG
3841	AAAACT CACGTT AAGGGA TTTTGG TCATGA GATTAT CAAAAA GGATCT TCACCT AGATCC
3901	TTTTAA ATTAAA AATGAA GTTTTA AATCAA TCTAAA GTATAT ATGAGT AAACTT GGTCTG
3961	ACAGTT ACCAAT GCTTAA TCAGTG AGGCAC CTATCT CAGCGA TCTGTC TATTTC GTTCAT
4021	CCATAG TTGCCT GACTCC CCGTCG TGTAGA TAACTA CGATAC GGGAGG GCTTAC CATCTG
4081	GCCCCA GTGCTG CAATGA TACCGC GAGACC CACGCT CACCGG CTCCAG ATTTAT CAGCAA
4141	TAAACC AGCCAG CCGGAA GGGCCG AGCGCA GAAGTG GTCCTG CAACTT TATCCG CCTCCA
4201	TCCAGT CTATTA ATTGTT GCCGGG AAGCTA GAGTAA GTAGTT CGCCAG TTAATA GTTTGC
4261	GCAACG TTGTTG CCATTG CTACAG GCATCG TGGTGT CACGCT CGTCGT TTGGTA TGGCTT
4321	CATTCA GCTCCG GTTCCC AACGAT CAAGGC GAGTTA CATGAT CCCCCA TGTTGT GCAAAA
4381	AAGCGG TTAGCT CCTTCG GTCCTC CGATCG TTGTCA GAAGTA AGTTGG CCGCAG TGTTAT
4441	CACTCA TGGTTA TGGCAG CACTGC ATAATT CTCTTA CTGTCA TGCCAT CCGTAA GATGCT
4501	TTTCTG TGACTG GTGAGT ACTCAA CCAAGT CATTCT GAGAAT AGTGTA TGCGGC GACCGA
4561	GTTGCT CTTGCC CGGCGT CAACAC GGGATA ATACCG CGCCAC ATAGCA GAACTT TAAAAG
4621	TGCTCA TCATTG GAAAAC GTTCTT CGGGGC GAAAAC TCTCAA GGATCT TACCGC TGTTGA
4681	GATCCA GTTCGA TGTAAC CCACTC GTGCAC CCAACT GATCTT CAGCAT CTTTTA CTTTCA
4741	CCAGCG TTTCTG GGTGAG CAAAAA CAGGAA GGCAAA ATGCCG CAAAAA AGGGAA TAAGGG
4801	CGACAC GGAAAT GTTGAA TACTCA TACTCT TCCTTT TTCAAT ATTATT GAAGCA TTTATC
4861	AGGGTT ATTGTC TCATGA GCGGAT ACATAT TTGAAT GTATTT AGAAAA ATAAAC AAATAG
4921	GGGTTC CGCGCA CATTTC CCCGAA AAGTGC CACCTG ACGTCT AAGAGA CCATTA TTATCA
4981	TGACAT TAACCT ATAAAA ATAGGC GTATCA CGAGGC CCTTTC GTCTTC AAGAAT TGTCTA
5041	GAGGCG CGCCGT TTAAAC CCTCAG CTGATC ATCCGG ATGTAC AGCGCG CGGCCG GCCGGT
5101	ACCACG CGTTGG CCACAT ATGGCG GCCGCT CGCGAT TAATTA ATCGCG ATGGCC ACATAT
5161	GGAGCT CTCTAG AGCTTG TCGACA GATCCC CCTCTT CATTTC TTTATG TTTTAA ATGCAC
5221	TGACCT CCCACA TTCCCT TTTTAG TAAAAT ATTCAG AAATAA TTTAAA TACATC ATTGCA
5281	ATGAAA ATAAAT GTTTTT TATTAG GCAGAA TCCAGA TGCTCA AGGCCC TTCATA ATATCC
5341	CCCAGT TTAGTA GTTGGA CTTAGG GAACAA AGGAAC CTTTAA TAGAAA TTGGAC AGCAAG
{tilde under (B)}{tilde under (a)}{tilde under (m)}{tilde under (H)}{tilde under (I)}
5401	AAAGCG AGGGGG ATCTGG ATCCTC CGGAGG GCCCCT TCTCCC TCTAAC ACTCTC CCCTGT
5461	TGAAGC TCTTTG TGACGG GCGAGC TCAGGC CCTGAT GGGTGA CTTCGC AGGCGT AGACTT
5521	TGTGTT TCTCGT AGTCTG CTTTGC TCAGCG TCAGGG TGCTGC TGAGGC TGTAGG TGCTGT
5581	CCTTGC TGTCCT GCTCTG TGACAC TCTCCT GGGAGT TACCCG ATTGGA GGGCGT TATCCA
5641	CCTTCC ACTGTA CTTTGG CCTCTC TGGGAT AGAAGT TATTCA GCAGGC ACACAA CAGAGG
5701	CAGTTC CAGATT TCAACT GCTCAT CAGATG GCGGGA AGATGA AGACAG ATGGTG CAGCCA
5761	CCGTAC GTTTGA TTTCCA CCTTGG TCCCCT GTCCAA AGGTGT AGGGTG TGTAAT AGCTCT
5821	GCTGAC AGTAGT ACACGC CCACAT CTTCGG CCTCCA CCCGGC TGATCT TCAGAG TGAAAT
5881	CTGTCC CAGATC CGCTGC CGCTGA ACCTGT CTGGCA CCCCGC TCTGCC GGGTGC TGGTCC
5941	AATAGA TCAGCA GCTGAG GGCTCT GCCCTG GTTTCT GCAGAT ACCAGG CCAGGT AGTTCT
6001	TCTGGT TCTCGC TGAACA GCAGGC TCTGGC TGCTCT TGCAGC TGATGC TGGCTG GCTCTC
6061	CGGGTG TCACAG GCAGGG ACAGTG GAGACT GGGTCA TCACGA TATCAC ATCTCA TGGCTG
{tilde under (E)}{tilde under (c)}oRI
6121	GCAGGA ACAGCA CCAGCA GCCCCA GCAGCT GCACTG GAGCCA TGGTGG CGGCGC TAGCGA
{tilde under (E)}{tilde under (c)}{tilde under (o)}{tilde under (R)}{tilde under (I)}
6181	ATTCTT AAGCCT GTGGAG AGAAAG GAACAG AAAACG AAACAA AGACGT AGAGTT GAGCAA
6241	GCAGGG TCAGGC AAAGCG TGGAGA GCCGGC TGAGTC TAGGTA GGCTCC AAGGGA GCGCCG
6301	GACAAA GGCCCG GTCTCG ACCTGA GCTTTA AACTTA CCTGTG GCCACA CGTGCA ATTGCT
6361	ATAGTG AGTCGT ATTAAT TTCGAT AAGCCA GTAAGC AGTGGG TTCTCT AGTTAG CCAGAG
6421	AGCTCT GCTTAT ATAGAC CTCCCA CCGTAC ACGCCT ACCGCC CATTTG CGTCAA TGGGGC
6481	GGAGTT GTTACG ACATTT TGGAAA GTCCCG TTGATT TTGGTG CCAAAA CAAACT CCCATT
6541	GACGTC AATGGG GTGGAG ACTTGG AAATCC CCGTGA GTCAAA CCGCTA TCCACG CCCATT
6601	GATGTA CTGCCA AAACCG CATCAC CATGGT AATAGC GATGAC TAATAC GTAGAT GTACTG
6661	CCAAGT AGGAAA GTCCCA TAAGGT CATGTA CTGGGC ATAATG CCAGGC GGGCCA TTTACC
6721	GTCATT GACGTC AATAGG GGGCGT ACTTGG CATATG ATACAC TTGATG TACTGC CAAGTG
6781	GGCAGT TTACCG TAAATA GTCCAC CCATTG ACGTCA ATGGAA AGTCCC TATTGG CGTTAC
6841	TATGGG AACATA CGTCAT TATTGA CGTCAA TGGGCG GGGGTC GTTGGG CGGTCA GCCAGG
6901	CGGGCC ATTTAC CGTAAG TTATGT AACGCG GAACTC CATATA TGGGCT ATGAAC TAATGA
6961	CCCCGT AATTGA TTACTA TTAATA ACTAGT CAATAA TCAATG TCAACG CGTATA TCTGGC
7021	CCGTAC ATCGGT AACTAG TCGGAC CGGCCC GGGCCA CCGGTG CTCGAA GCTTGG ATCGAT
7081	CCAGAC ATGATA AGATAC ATTGAT GAGTTT GGACAA ACCACA ACTAGA ATGCAG TGAAAA
7141	AAATGC TTTATT TGTGAA ATTTGT GATGCT ATTGCT TTATTT GTAACC ATTATA AGCTGC
7201	AATAAA CAAGTT AACAAC AACAAT TGCATT CATTTT ATGTTT CAGGTT CAGGGG GAGGTG
7261	TGGGAG GTTTTT TAAAGC AAGTAA AACCTC TACAAA TGTGGT ATGGCT GATTAT GATCTC
7321	TAGTCA AG

[0086] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0087] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.