Thermostable flap endonuclease derived from a hyperthermophile bacterium belonging to the genus pyrococcus

[0002] 1. Field of the Invention

[0003] The present invention relates to a thermostable Flap endonuclease effective for genetic recombination and genetic shuffling based on low homology, as well as to a gene thereof.

[0004] 2. Description of the Prior Art

[0005] There are a method for random mutation of a broad region in a gene and a method for random mutation of a local region in a gene. In the former method, polymerase chain reaction (PCR) is applied such that a specific nucleotide is deleted to induce mutation at the time of replication of the target gene, and in the latter method, PCR using mixed primers is applied to mutate the target site. However, there is still no method for highly efficient induction of genetic recombination or genetic shuffling based on low homology in vitro. A speculative mechanism of genetic recombination and genetic shuffling in vivo is shown in FIG. 1. Step 1 of FIG. 1 shows formation of a single-stranded overhang by a 3′-5′ exonuclease; step 2 shows formation of temporary nucleotide base pairs based on low homology; step 3 shows repair of the gap by DNA polymerase and formation of a Flap structure; step 4 shows removal of the Flap single strand by a Flap endonuclease; and step 5 shows ligation of nicks by a DNA ligase. However, the properties of the enzymes catalyzing the respective steps are not fully elucidated. The Flap endonuclease is an enzyme catalyzing step 4. As shown in FIG. 2, the Flap endonuclease specifically recognizes the Flap structure in DNA and cleaves the single strand (called Flap), and this enzyme is found in mammalian cells and yeast cells. Since its origin is organisms living at normal temperatures, this enzyme has poor thermostability and thus is not suitable for artificial genetic shuffling reaction, including PCR.

[0006] Because the conventional Flap endonuclease is unstable at high temperatures, it cannot be used to develop methods where genetic recombination or genetic shuffling based on low homology in vitro is induced at high temperatures. However, if a thermostable Flap endonuclease functioning stably at high temperatures can be obtained, it becomes possible to develop new techniques of conducting artificial homologous recombination or genetic shuffling highly efficiently by coupling the enzyme reaction with PCR. Accordingly, development of the thermostable Flap endonuclease stable at high temperature has long been desired.

[0007] Under these circumstances, the object of the present invention is to provide a novel thermostable Flap endonuclease and a gene thereof.

[0008] To achieve this object, the present inventors directed their attention to a hyperthermophile bacterium growing at 90 to 100° C., and as a result, a gene presumed to bring about the activity of the present enzyme was found in a gene sequence from the bacterium. Further, the enzyme was produced in E. coliby use of said gene, and it was confirmed that this enzyme is stable at high temperatures (75° C. or more) and exhibits structurally specific endonuclease activity. The present invention was thereby completed.

[0009] That is, the present invention relates to a thermostable Flap endonuclease whose optimum temperature is 75° C. or more.

[0010] Further, the present invention relates to a DNA coding for the following protein (a) or (b):

[0011] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO:2; and

[0012] (b) a protein with Flap endonuclease activity, consisting of an amino acid sequence where in the amino acid sequence (a), one or more amino acids are deleted, substituted or added.

[0013] The DNA is a DNA specifically shown in SEQ ID NO:1.

[0014] The addition, deletion or substitution of amino acids can be effected using site-directed mutagenesis known in the art (see e.g. Nucleic Acid Research, Vol. 10, No. 20, pp. 6487-6500 (1982)). The number of one or more amino acids added, deleted or substituted is the number of amino acids which can be added, deleted or substituted by site-directed mutagenesis.

[0015] Further, the present invention relates to a recombinant vector comprising said DNA.

[0016] Further, the present invention relates to a transformant transformed with the recombinant vector comprising said DNA.

[0017] Further, the present invention relates to a process for producing a thermostable Flap endonuclease, which comprises culturing said transformant in a medium to produce the thermostable Flap endonuclease.

[0018] FIG. 1 shows a mechanism of homogeneous recombination and genetic shuffling in vivo in which the thermostable Flap endonuclease is involved. The Flap endonuclease catalyzes step 4.

[0019] FIG. 2 shows one example of the DNA structure (SEQ ID NOS:5, 6 & 7, respectively) which can serve as a substrate for the Flap endonuclease. The single-stranded portion is called a Flap. The arrow indicates the site to be cleaved by this enzyme.

[0020] Hereinafter, the present invention is described in detail.

[0021] The hyperthermophilic bacterium used in the present invention is sulfur-metabolizing thermophilic archaebacterium, Pyrococcus horikoshiiJCM9974.

[0022] The thermostable Flap endonuclease of the present invention was obtained in the following manner. PCR was applied using chromosomal DNA as a template from Pyrococcus horikoshiiJCM9974 and the upper and lower primers shown in Example 6, and a DNA fragment containing a Flap endonuclease gene was isolated.

[0023] This gene was inserted into protein expression plasmid pET 15b, then the resulting recombinant plasmid was integrated in E. coli,and the transformed E. coliwas cultured in a medium to produce the present enzyme. The produced thermostable Flap endonuclease was isolated and purified by heat treatment and column chromatography. It was confirmed that the purified Flap endonuclease is a protein with a molecular weight of about 40,000 and is an enzyme which recognizes the Flap type DNA substrate shown in FIG. 2 and hydrolyzes a single-stranded portion (Flap portion) in an oligonucleotide.

[0024] The activity of the present enzyme did not decrease even after treatment of the enzyme at 95° C. for several hours in 50 mM phosphate buffer, pH 7.5 containing 1 M NaCl. Further, the optimum pH for activity was 6.0 to 8.0, and the optimum temperature was about 75° C. or more at pH 8.

[0025] According to the present invention, there is provided a thermostable Flap endonuclease whose optimum temperature for reaction is 75° C. or more. Further, the present enzyme is thermally stable, so it becomes possible to develop new techniques of conducting artificial homologous recombination and genetic shuffling highly efficiently by coupling the enzyme reaction with PCR.

[0026] Hereinafter, the present invention is described in detail with reference to Examples, which, however, are not intended to limit the technical scope of the present invention.

[0027] Pyrococcus horikoshiiJCM9974 was cultured in the following manner.

[0028] Dissolved in 1 L were 13.5 g sodium chloride, 4 g Na₂SO₄, 0.7 g KCl, 0.2 g NaHCO₃, 0.1 g KBr, 30 mg H₃BO₃, 10 g MgCl₂·6H₂O, 1.5 g CaCl₂, 25 mg SrCl₂, 1.0 ml resazurin solution (0.2 g/L), 1.0 g yeast extract and 5 g Bacto-trypton, and this solution was adjusted to pH 6.8 and sterilized under pressure. Then, dry-sterilized elementary sulfur was added thereto at a concentration of 0.2%, and this medium was rendered anaerobic by saturation with argon, and Pyrococcus horikoshiiJCM9974 was inoculated thereinto. Whether the medium became anaerobic or not was confirmed by failure of Na₂S to cause the resazurin to turn pink in the culture solution upon addition of a Na₂S solution. This culture solution was incubated at 95° C. for 2 to 4 days, and the resulting culture was harvested by centrifugation at 5000 rpm for 10 minutes to give 1 g of the microorganism.

[0029] Chromosomal DNA from Pyrococcus horikoshiiJCM9974 was prepared in the following manner. The microorganism obtained (0.1 g) in Example 1 was washed twice with 10 mM Tris buffer (pH 7.5), 1 mM EDTA and enclosed in an InCert Agarose (FMC Co., Ltd.) block. This block was treated with 1% N-lauroylsarcosine and 1 mg/ml protease K, whereby the chromosomal DNA was separated and prepared in the Agarose block.

[0030] The chromosomal DNA obtained in Example 2 was partially digested with restriction enzyme HindIII, and then a fragment of about 40-kb in length was prepared by agarose gel electrophoresis. This DNA fragment was ligated by T4 ligase to Bac vector pBAC108L (Ung-Jon Kim et al., Nucleic Acid Research, 20(5), 1083-1085 (1992)) and pFOSI (Ung-Jon Kim et al., Nucleic Acid Research, 20(5), 1083-1085 (1992)), both vectors having been completely digested with restriction enzyme HindII. If the former vector was used, the DNA after ligation was immediately introduced by electroporation into E. coli.If the latter vector pFOS1 was used, the DNA after ligation was packaged into λ-phage particles in vitro by GIGA Pack Gold (Stratagene) and these particles were infected into E. coliwhereby the DNA was introduced into the E. coli.The resulting E. colipopulations resistant to antibiotic chloramphenicol were used as BAC and Fosmid libraries respectively. From the libraries, clones suitable for covering the chromosome of Pyrococcus horikoshiiJCM9974 were selected and alignment of the clones was conducted.

[0031] Nucleotide sequencing of each of the aligned BAC and Fosmid clones was conducted in the following manner. The BAC or Fosmid clone DNA recovered from the E. coliwas fragmented by ultrasonication, and 1- and 2-kb long DNA fragments were recovered by agarose gel electrophoresis. Shotgun clones with the fragments inserted into a HincII restriction enzyme site on plasmid vector pUC118, that is, 500 clones derived from each of the BAC and Fosmid clones, were prepared. The nucleotide sequence of each shotgun clone was determined by Automatic Nucleotide Sequence Reader 373 or 377 manufactured by Perkin Elmer, ABI. The nucleotide sequences obtained from the respective shotgun clones were linked and compiled by nucleotide sequence automatic linking software Sequencer to determine the whole nucleotide sequence of each of the BAC and Fosmid clones.

[0032] The nucleotide sequence of each of the BAC and Fosmid clones determined above was analyzed by a large computer to identify a gene coding for the Flap endonuclease. The sequence of this gene is shown in SEQ ID NO:1. The amino acid sequence deduced from this nucleotide sequence is shown in SEQ ID NO:2.

[0033] For the purpose of creating restriction enzyme sites (NdeI and XhoI) upstream and downstream from the structural gene, DNA primers were synthesized, and by PCR, the restriction enzyme sites were created in regions upstream and downstream from the gene.

[0034] Upper primer (SEQ ID NO:3): 5′-GGGAATTCCTGCAGATCGCATATGGGTGTTCCTATCGGTGAC-3′

[0035] Lower primer (SEQ ID NO:4): 5′-ACTAATCCCGGGTACCTCGAGGCTATAGACTTTAGGGTTTCT-3′

[0036] After PCR, the product was completely digested (37° C., 2 hours) with the restriction enzymes (NdeI and XhoI) and the structural gene was then purified.

[0037] pET-15b (Novagen) was cleaved with restriction enzymes NdeI and XhoI, then purified and ligated by T4 ligase to the above structural gene at 16° C. for 2 hours. A part of the ligated DNA was introduced into E. coli-XL 1-Blue MRF1 competent cells to give a transformant colony. The expression plasmid was purified from the resulting colony by the alkali method (Sambrook, J. et al., Molecular Cloning: A laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory (1989)).

[0038] E. colicompetent cells (E. coliBL21 (DE3) available from Novagen) were fused and 0.1 ml cells were transferred to a Falcon tube. The expression plasmid solution (0.005 mL) was added to the cells, left on ice for 30 minutes, and heat-shocked at 42° C. for 30 seconds, and 0.9 mL SOC medium was added thereto, and the cells were cultured at 37° C. for 1 hour under shaking. Then, the cells were plated on a 2YT agar plate containing ampicillin and cultured at 37° C. overnight to give a transformant. This transformant has been deposited as FERM P-16389 on Aug. 18, 1997 with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan.

[0039] This transformant was cultured in ampicillin-containing 2YT medium, 2L, until its absorbance at 600 nm reached 1, and then IPTG (isopropyl-β-D-thiogalactopyranoside) was added thereto, followed by culturing for further 6 hours. After culturing, the transformant was harvested by centrifugation (6,000 rpm, 20 minutes) to give 30 g of the microorganism.

[0040] The harvested microorganism (30 g)was frozen at −20° C. and thawed, then alumina in an amount as twice as that of the microorganism and 1 mg DNase were added thereto, and the microorganism was disrupted, followed by adding a 5-fold excess amount of 10 mM Tris-HCl buffer (pH 8.0) to give a suspension. The resulting suspension was heated at 85° C. for 30 minutes and centrifuged (11,000 rpm, 20 minutes), and the supernatant was subjected to affinity chromatography on a Ni column (using Novagen, His-Bind metal chelation resin & His-Bind buffer kit). A fraction eluted with 60 mM imidazole was adsorbed onto a HiTrap SP (Pharmacia) and eluted with a concentration gradient of NaCl to give an active fraction. The resulting active fraction solution was further applied to a Hitrap Heparin column (Pharmacia) and eluted with a concentration gradient of NaCl to give 1 mg purified enzyme.

[0041] (1) Synthetic oligonucleotides

[0042] All oligonucleotides were synthesized by Grainer Japan Co., Ltd. The names and sequences of the respective oligonucleotides are as follows.

[0043] F_brstrand (SEQ ID NO:5): 5′-GGACTCTGCCTCAAGACGGTAGTCAACGTG-3′

[0044] F_adjstrand (SEQ ID NO:6): 3′-CTGCCATCAGTTGCAC-5′

[0045] Flap strand (Seq ID NO:7): 3′-CCTGAGACGGAGTTTCAATCCTGACGAACTGTAG-5′

[0046] (2) Preparation of the Flap substrate (FIG. 2)

[0047] The Flap strand was labeled at the 5′-terminal thereof with ³²P by means of [γ-³²P]ATP and with T₄polynucleotide kinase. The labeled Flap strand was boiled together with the F_brand F_adjstrands in 20 mM Tris-HCl buffer, pH 7.4, 150 mM NaCl and then annealed by gradually lowering the temperature of the solution to 4° C.

[0048] (3) Flap endonuclease activity

[0049] The ³²P-labeled Flap substrate and the enzyme (400 fmol) were added to 15 ml of 50 mM Tris-HCl buffer (pH 8, 1.5 mM MgCl₂, 0.5 mM β-mercaptoethanol, 100 mg/ml bovine serum albumin) and reacted at 50° C. for 30 minutes. Then, 15 ml of 95% formamide, 10 mM EDTA and 1 mg/ml xylene cyanol were added to stop the enzyme reaction. Further, this reaction solution was heated at 95° C. for 5 minutes and electrophoresed at 2000 V for 2 hours in 15% polyacrylamide gel (16×45 cm) containing 7 M urea and 1×TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8). The reaction product was imaged and quantified by Molecular Imager GS-525 (Bio-Rad). One unit activity is defined as the amount of the enzyme causing 1 fmol Flap substrate to be decomposed under usual conditions for measuring Flap endonuclease activity.

[0050] Properties of the enzyme

[0051] (1) Substrate specificity

[0052] The present enzyme does not act on double-stranded DNA or single-stranded DNA and has the endonuclease activity of cleaving off the single-stranded DNA (Flap) from the DNA structure shown in FIG. 2.

[0053] In the substrate shown in FIG. 2, the arrow indicates the cleavage site from which the 20-mer single-stranded DNA is released. Further, the activity of this enzyme does not depend on the nucleotide sequence of DNA.

[0054] (2) Optimum pH

[0055] The optimum pH for enzyme activity was determined by measuring the initial rate of hydrolysis of the ³²P-labeled Flap substrate (FIG. 2) by the enzyme at 50° C. in 50 mM sodium acetate buffer, 50 mM phosphate buffer and 50 mM borate buffer (pH 4 to 9), respectively. Because the maximum initial rate was achieved in the vicinity of pH 6.0 to 8.0, it was concluded that the optimum pH was 6.0 to 8.0.

[0056] (3) Optimum temperature

[0057] A predetermined amount of the enzyme was added to 50 mM phosphate buffer, pH 8.0 containing 400 fmol ³²P-labeled Flap substrate (FIG. 2) as the substrate and reacted for 30 minutes to examine its relative activity. The maximum activity (optimum temperature) was 75° C. or more.

[0058] (4) Thermostability

[0059] An aqueous solution containing the enzyme at a concentration of 0.1 mg/ml (50 mM phosphate buffer, pH 8.0, 1 M NaCl) was heated at 95° C. for 5 hours and then examined for its residual activity. The result indicated that the half-life of the activity was 5 hours. Further, a differential scanning colorimeter (DSC) was used to examine thermal denaturation. When 0.5 mg/ml of the enzyme solution (50 mM phosphate buffer, pH 8.0, 1 M NaCl) was measured at 0 to 125° C., it was revealed that the thermal denaturation temperature (Tm) is 103° C.