WO1998049274A1 - Thermostable dna polymerase and inteins of the thermococcus fumicolans species - Google Patents

Thermostable dna polymerase and inteins of the thermococcus fumicolans species Download PDF

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WO1998049274A1
WO1998049274A1 PCT/FR1997/000761 FR9700761W WO9849274A1 WO 1998049274 A1 WO1998049274 A1 WO 1998049274A1 FR 9700761 W FR9700761 W FR 9700761W WO 9849274 A1 WO9849274 A1 WO 9849274A1
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Joël QUERELLOU
Marie-Anne Cambon
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Appligene-Oncor
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Priority to PCT/FR1997/000761 priority Critical patent/WO1998049274A1/en
Priority to PCT/FR1998/000868 priority patent/WO1998049275A2/en
Publication of WO1998049274A1 publication Critical patent/WO1998049274A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the present invention relates to a new thermostable DNA polymerase and its two inteins, originating from an archaebacterium of the species Thermococcus fumicolans.
  • DNA polymerases are enzymes involved in the replication and repair of DNA in any living cell. Many DNA polymerases isolated from microorganisms such as E. coli are known today.
  • DNA polymerase I DNA polymerase I
  • phage T4 DNA polymerases have also been identified and purified and from thermophilic microorganisms such as Thermus aqua ticus (Taq polymerase, Chien, A. et a. J. Bact. 1976,
  • Thermus thermophilus or species of the genus Bacillus (European patent application published under No. 699 760), Thermococcus (European patent application No. 455 430), Sulfolobus and Pyrococcus (patent application
  • DNA polymerases The mechanism of action of DNA polymerases is relatively well known today and consists of replication of DNA identically according to a semi-conservative mode. The copied strand serves as a matrix and the four nucleotide triphosphates are the substrate for this polymerization. Enzymes with DNA polymerase activity are increasingly used today in vitro to work in molecular biology for various purposes such as cloning, error detection,
  • This amplification, in vi tro, of deoxyribonucleic acid sequences calls upon the technique of the polymerase chain reaction (PCR) described in European patents Nos. 200 362 and 201 184.
  • the principle of this technique is based on the carrying out successive cycles of extension of primers using the four nucleotide triphosphates as well as a DNA polymerase and a template DNA to be copied.
  • the enzyme doubles the number of DNA strands available and between each cycle thermodenaturation is necessary in order to open the DNA double helix for the next cycle.
  • the temperatures used for this thermodenaturation step are not compatible with the conservation of the activity of most of the known DNA polymerases, such as Klenow.
  • thermophilic microorganisms are known today, it still remains difficult to obtain these thermostable enzymes with sufficient production yields.
  • Molecular biology and genetic engineering overcome this drawback.
  • the gene coding for DNA polymerase is cloned, sequenced and then recloned in an expression vector in order to produce the so-called recombinant protein, in a mesophilic host which is easier to cultivate such as E. coli or S. cerevisiae.
  • This expression method in E. coli has in particular been described in the international patent application PCT published under No. WO 89/06691 for producing DNA polymerase from Thermus aquaticus.
  • the DNA polymerase of the invention comes from an archaebacterium of the species Thermococcus fumicolans. In addition to its thermostable properties making it particularly effective in particular in a PCR process, this DNA
  • ISAEP polymerase is remarkable in that it has two "protein introns”, also called “inteins”, at the level of its precursor polypeptide.
  • the nucleotide sequence of its inteins is inserted into that of DNA polymerase, generally at the level of conserved sites involved, after translation, in catalytic reactions. These sequences are transcribed and translated at the same time as that of DNA polymerase and the autocatalytic splicing of the inteins then produces three enzymes: two inteins and one DNA polymerase.
  • inteins are also found in other molecules such as vacuolar ATPase in S. cerevisiae (4), GyrA in Mycobacterium leprae (7), Rec A in Mycobacterium tuberculosis (5, 6).
  • the inteins belong for the most part to the family of endonucleases of the "homing endonucleases” type since they cut DNA at a recognized site, at the very place where their nucleotide sequence is inserted.
  • the development of biotechnologies both in research and in the fields of medicine or agri-food requires having various types of DNA polymerases capable of improving quantitatively and qualitatively techniques as diverse as cloning, detection , amplification of DNA sequences.
  • the present invention aims precisely to offer a new thermostable DNA polymerase which is derived from a recently described species: Thermococcus fumi colans (8).
  • This isolate was isolated from fragments of chimneys taken in the North Fidgian basin during the Franco-Japanese STARMER campaign in 1989.
  • This species strict anaerobic, has an optimal growth temperature of 90 ° C, which is relatively high for a Thermococcus. Its optimum pH is 8.8, and its salinity level from 20 g / 1 to 40 g / 1.
  • the subject of the invention is therefore a purified thermostable DNA polymerase of archaebacteria of
  • the invention therefore relates to purified thermostable DNA polymerase of archaebacteria of the species Thermococcus fumicolans having a molecular weight of the order of 89,000 daltons as well as its enzymatically equivalent derivatives.
  • the term “enzymatically equivalent derivatives” is understood to mean the polypeptides and proteins constituted by or comprising the amino acid sequence represented in the sequence list in the appendix under the number SEQ ID NO: 2 as soon as they exhibit the properties of the DNA polymerase of Thermococcus fumi col ans.
  • the invention more particularly contemplates a DNA polymerase, the amino acid sequence of which is represented in the sequence list in the appendix under the number SEQ ID NO: 1 or a fragment thereof or an assembly of such fragments, such as the 774 amino acid sequence represented in the sequence list in the appendix under the number SEQ ID NO: 2.
  • derivatives is also understood to mean the amino acid sequences modified above by insertion and / or deletion and / or substitution of one or more amino acids, provided that the properties of the DNA polymerase of T. fumicolans which result therefrom are not significantly changed.
  • the invention also relates to a DNA sequence consisting of or comprising the sequence coding for a DNA polymerase of the invention.
  • the DNA sequence represented in the annexed sequence list under the number SED ID NO: 1 represents such a sequence.
  • the DNA coding for the DNA polymerase of T. fumicolans and its two inteins consists of nucleotides 357 to 5028. Nucleotides 1 to 356 correspond to the promoter region of this gene. Consequently, the subject of the invention is a DNA sequence constituted by or comprising the sequence between nucleotides 357 to 5028 of SED ID NO: 1, or a fragment thereof, or an assembly of such fragmen s .
  • the invention relates more particularly to a DNA sequence constituted by or comprising nucleotides 357 to 1674 and 2755 to 3156 and 4324 to 5028 of the DNA sequence represented in the sequence list in the appendix under the number SED ID NO : 1.
  • This sequence codes for the DNA polymerase of T. fumicolans whose sequence of 774 amino acids is represented in the sequence list in the appendix under the number SED ID NO: 2.
  • the invention relates as much to DNA polymerase isolated and purified from the Thermococcus fumicolans strain as to DNA polymerase prepared by chemical synthesis, for example by ligation of polypeptide fragments, or also by genetic engineering methods. Within the framework of these genetic engineering methods, the invention also relates to a vector comprising a DNA sequence defined above, as well as a process for the production or expression in a cellular host of the thermostable DNAs of the invention. .
  • a process for the production of a DNA polymerase according to the invention consists in:
  • the cell host used in the above methods can be chosen from prokaryotes or eukaryotes and in particular from bacteria, yeasts, mammalian, plant or insect cells.
  • the vector used is chosen according to the host to which it will be transferred.
  • thermostable DNA polymerase of the invention is useful in particular in the methods of enzymatic amplification of nucleic acid sequences. Consequently, the subject of the invention is such methods using the thermostable DNA polymerase described above, as well as the amplification kits comprising, in addition to the reagents generally used, an adequate quantity of this DNA polymerase.
  • the invention also relates to a purified thermostable intein of archaebacteria of the species Thermococcus fumicolans.
  • the inteins are also defined as protein introns which are not spliced at the level of the messenger RNA but at the level
  • RECTIFIED SHEET (RULE 91) ISA / EP protein maturation. They therefore relate to a single gene translated and transcribed in a single step, and constitute by-products of the maturation of the protein encoded by this gene (Xu, MG, Comb, DG., Paulus H., Noren CJ, Shao Y ., Perler, F., 1994, Protein splicing: an analysis of the branched intermediate and its resolution by succinimidine formation. EMBO J. 13, 5517-5522.)
  • the inteins are restriction endonucleases which have the property of cutting DNA at the very place where their gene is inserted, and therefore they can be considered as selfish sequences.
  • the inteins have in their sequence all the information necessary for their own splicing since they splice in E. coli.
  • a first step in the formation of a linear intermediary which has an ester function is a first step in the formation of a linear intermediary which has an ester function.
  • This reaction is dependent on the pH and the local environment of this bond (nature of the amino acids).
  • This principle is used in cloning, expression or purification kits using inteins, because a change in the environment causes splicing or not. Indeed, it would be inhibited at pH 11 and activated at pH 7.5.
  • the third step consists in a cyclization of 1 asparagine releasing the intein.
  • the fourth step is the stabilization of the mature protein and the formation of a real peptide bond.
  • thermosensitive mutants making it possible to block splicing.
  • This possibility of controlling protein splicing by temperature can be used in cloning vectors with a sequence coding for the intein and around cloning sites. If the protein to be cloned and expressed is toxic to the host, it can be cloned into two fragments around the intein sequence. Thus, overall, the gene to be cloned is complete but it is interrupted by the sequence of the intein. During expression, the intein is found in the expressed protein, thus making it inactive. It is then possible by heating, at the end of the induction, to release the intein by autocatalytic splicing and thus find the cloned active protein.
  • the inteins thus make it possible to carry out purifications and are used in kits according to the principle described below. Certain residues around the intein splicing site are modified.
  • the expression of the recombinant protein is carried out at low temperature to block possible splicing too early.
  • At the C-terminal of the intein is fixed a site having a strong affinity for chitin.
  • the cloned protein is expressed as well as the intein and the chitin binding site.
  • the purification is then carried out with the chitin, on affinity columns, which retain the chitin and also the intein and the cloned protein, the whole being part of the same pre-protein.
  • the N-terminal end of the intein is then hydrolyzed with DTT or ⁇ -mercaptoethanol to release the cloned protein.
  • thermostable restriction endonucleases which have as a recognition site the very place where their gene is inserted in the "host” sequence. They have a twice repeated nucleic sequence (LAGLIDADG) in the protein,
  • RECTIFIED SHEET (RULE 91) ISA / EP more or less conserved sequence which corresponds to the active DNA recognition and cleavage site. These enzymes also seem to need g ++ for their activity. It should be noted that the two inteins of the invention are co-expressed in E. coli and are self-splicing. This means that they have no toxicity for the host, unlike one of the inteins of Thermococcus li toralis (9), and therefore their use in expression or purification kits is easy. .
  • a first intein sequence according to the invention is represented in the annexed sequence list under the number SEQ ID NO: 3.
  • This intein, called I-Tfu-1 has a molecular weight of 41,409 Da and a pi of 9.13, deduced from the 360 amino acid sequence of the sequence SEQ ID NO: 3.
  • a second intein sequence according to the invention is represented in the annexed sequence list under the number SEQ ID NO: 4.
  • This intein called I-Tfu-2, has a molecular weight of 44,765 Da and pi of 9 , 6, deduced from the 389 amino acid sequence of the sequence SEQ ID NO: 4.
  • thermostable inteins of the invention are useful in particular in methods of restriction of nucleic acids and in the development of expression vectors making it possible to reduce the toxicity of the protein to be expressed by inserting one of the two sequences of inteins in the sequence of the protein to be expressed. This can be done without manipulation of the sequence of inteins if the cloning is carried out in E. coli, the expression techniques used having demonstrated their harmlessness for this host organism. Consequently, the subject of the invention is such processes using one or both of the two thermostable inteins described above, as well as the expression or purification kits containing one or the two sequences coding for said thermostable inteins.
  • the invention also relates to a DNA sequence consisting of or comprising the sequence coding for an intein of the invention.
  • nucleotides 1675 and 2754 in the sequence SED ID NO: 1 in the appendix This DNA sequence codes for the intein, the amino acid sequence of which is represented in the annexed sequence list under the number SED ID NO: 3.
  • the invention relates both to these thermostable inteins isolated and purified from the Thermococcus fumicolans strain as to inteins prepared by chemical synthesis, for example by ligation of polypeptide fragments, or also by genetic engineering methods.
  • the invention also relates to a vector comprising a DNA sequence defined above, as well as a process for the production or expression in a cellular host of DNA coding for the inteins of the invention. Such methods are identical to those reported previously for T DNA polymerase. fumicolans.
  • thermostable DNA of the invention RECTIFIED SHEET (RULE 91) ISA / EP of the thermostable DNA of the invention, and referring to the accompanying drawings in which:
  • FIG. 1 shows the DNA-DNA hybridization of the genomic DNA of T. fumicolans digested with various restriction enzymes and hybridized with the probes GE23ClaI-HindIII and GE23XhoI-SalI.
  • FIG. 2 shows the cloning strategy, the gene structure of the DNA polymerase of T. fumiculans and the gene products.
  • FIG. 3 shows the results of purification of the recombinant polymerase of T. fumi colans after a heparin sepharose column, visualized by SDS-PAGE.
  • FIG. 4 represents the results of purification of the recombinant polymerase of T. fumiculans after a Blue column HTrap n ° 2, visualized by SDS-PAGE.
  • FIG. 5 shows the purification results of the recombinant polymerase of T. fumi col years after a phosphocellulose column, visualized by SDS-PAGE.
  • FIG. 6 represents the results of purification of the recombinant polymerase of T. fumicolans after a MonoQ column, visualized by SDS-PAGE.
  • FIG. 7 shows the PCR results with DNA polymerase from T. fumicolans with the exclusion fractions from the MonoQ column.
  • the strain Pyrococcus sp. GE 23 was isolated from chimneys of deep hydrothermal vents and was supplied by G Erauso (CNRS, Station Bitician de Roscoff, France).
  • the Thermococcus fumicolans strain was obtained from the Marine Microbiology laboratory of G. Barbier (IFREMER-DRV-VP-CMM) in Brest, France.
  • This strain, Thermococcus fumicolans was obtained by purification from fragments of hydrothermal vents collected in the North Fidgian basin during the Franco-Aponais campaign STARMER carried out in 1989 at 2000 meters depth.
  • Pyrococcus sp.GE23 was cultured at 85 ° C in 2216S medium (DIFCO) at a pH of 6.5.
  • Thermococcus fumi col ans described in 1996 (Godfroy et al.) Is cultivated under anaerobic conditions in a medium containing the following elements: peptone 2g / 1; yeast extracts 0.5 g / l; sea salt (Sigma) 30g / l; PIPES buffer 6.05g / l, elemental sulfur 10g / 1, rezasurin lmg / 1. The pH is adjusted to 8.5 with 5N sodium hydroxide at 20 ° C.
  • the Escherichia coli SURE strain (Stratagene, La Jolla, Calif.) was cultured in LB medium with the appropriate antibiotic (s), at 37 ° C. with stirring. This strain was used as a host to receive the primary constructs from the pUC 18 or pBluescript vectors.
  • the NovaBlue, BL21 (DE3) and BL21 (DE3) pLysS strains (Novagen, Madison, Wi.) Were cultured in 2xYT medium with the appropriate antibiotics at 37 ° C or 30 ° C. These strains were used as hosts for the expression plasmids.
  • Thermococcus fumicolans was isolated by the modified Charbonnier method (3).
  • the cells were resuspended in TE-Na IX buffer, then lysed at 40 ° C for three hours
  • RECTIFIED SHEET (RULE 91) ISA / EP with a mixture of 1% N-Lauryl-sarcosine, 1% sodium dodecyl sulfate and 0.4 mg / ml of proteinase K. After lysis, centrifugation at 5000 g for 10 minutes makes it possible to remove cellular debris.
  • the DNA is extracted by a treatment with Phenol-Chloroform-Isoamyl alcohol or PCI (25-24-1), then treated with RNAse at 5 ⁇ g / ml at 60 ° C for one hour. These steps are followed by two additional PCI extractions and a chlorophoric extraction.
  • the DNA is then precipitated with absolute ethanol at -20 ° C, then centrifuged, air dried and taken up in TE-1x buffer. The concentration and purity of this DNA are measured by spectrophotometry at 230, 260 and 280nm with a GeneQuantlI device (Pharmacia, Upsalla, Sweden).
  • the DNA was completely digested overnight at 37 ° C. by a series of restriction enzymes (BamHI, HindIII, EcoRI, EcoRV, PvuII, Sali , Xbal and Xhol) by simple and double digestions.
  • DNA fragments are fractionated on 0.8% agarose gel in TBE-1X and transferred in vacuo to a Hybond-N + nylon membrane (Amersham, UK). These membranes were hybridized with DNA probes prepared by PCR with specific primers selected from the DNA polymerase genes of P. furi osus, T. li toral is and Pyrococcus sp. GE23. These probes are previously marked with 32P by "random primmg" in accordance with the manufacturer's recommendations (Megaprime, Amersham, UK).
  • Tli I and Tli T covering respectively the regions delimited by base pairs 297 to 1768 and 4631 to 5378 respectively, as defined by Hodges et al. (9).
  • Pyrococcus sp GE23 probes were used, one containing the 5 'part of the gene (Clal-HindIII fragment
  • RECTIFIED SHEET (RULE 91) ISA / EP corresponding to sites pb 8 and pb 1353 of the coding section) and the other containing the terminal part of this same gene obtained by PCR (primers known as expression Ndel and Sali corresponding to sites pb -1 and pb 2318, then digestion by Xhol and Sali including the bases of Nos. 1879 to 2318). Positive fragments were identified by DNA-DNA hybridization (14). Only hybridizations with Pyrococcus sp. GE 23 provided positive signals at 55 ° C in less than 24 h of exposure. The probes from T. li toralis and P. furiosus gave no results, even at 50 ° C in a standard buffer without formamide.
  • HindIII fragments of 1.9 kb were selected, then prepared by appropriate digestion of 100 ⁇ g of genomic DNA, purified in dialysis bags from agarose gels, and precipitated with absolute ethanol after PCI extraction. The fragments were ligated into a dephosphorylated pUC 18 / HindIII.
  • the transformations of the host strains were carried out by electroporation (Gene Inc., Biorad).
  • the screening of the recombinant clones was carried out by selection with ampicillin, alpha-complementation on X-Gal-IPTG substrate then hybridization of colonies according to standard techniques (12). The temperature of the colony hybridizations was 55 ° C.
  • the plasmid DNA was isolated according to the method described by Birnboim and Doly (1), then purified by anion exchange chromatography in solid phase (Quiagen, Chatsworth, Calif.). The restriction fragments of the plasmids were purified on agarose gel by the GeneClean method (Bio 101, La Jolla, Calif.) For later cloning.
  • Thermococcus fumicolans 16S and 23S rRNA was amplified by PCR using the following primers:
  • the PCR product was cloned into the vector pUC18 for subsequent sequencing.
  • the DNA sequences were obtained by the chain termination method (13) using an Applied Biosyste s automatic DNA analysis system.
  • the two strands of the genes coding for DNA polymerase and the two inteins were sequenced using universal primers localized on vectors as well as internal primers.
  • the 16S rDNA sequence was carried out on both strands, after cloning (SureClone, Pharmacia, Uppsala, Sweeden), using the Hot-Tub kit (Amersham, UK.) In order to remove the compressions.
  • PCR was used to prepare the complete fragment of DNA polymerase and the two inteins in using primers containing the NdeI and BamHI restriction sites:
  • the reaction mixture contained GOLDSTAR DNA polymerase (Eurogentec, B), the Taq Extender enzyme (containing Pfag from Stratagene), the extension buffer with the four dNTPs (each at 0.2mM) and the primers Tfu Dir and Tfu Rev at 50 pmol in a volume of 50 ⁇ l final.
  • the amplification was carried out over 20 cycles: 1 min 94 ° C, 1 min at 54 ° C and 6 min at 72 ° C using a Stratagene 96-gradient thermocycler.
  • the PCR fragments were then digested with the enzymes NdeI and BamHI and then ligated to the same sites of the vector, thus restoring the initiation codon.
  • the construction thus obtained was named PARHS2TFU1.
  • This construction was sequenced at the junction sites to verify its integrity with respect to the genomic DNA sequence.
  • the expression tests were carried out according to the following protocol: selection of the recombinant clones in the E. coli Novablue strain, expression with the BL21 (DE3) pLysS strain in a 2xYT medium and induction for four hours at ImM of IPTG.
  • the first induction tests were carried out on 5 ml cultures, whether induced or not. Night precultures are carried out without an inductor and restarted in a fresh medium in the morning (to the tenth), until the optical density (measured at 600nm) is 0.6, then either induced for 4 hours, or not induced and stopped after 2, 4 or 14 hours. Four ml of cultures are then centrifuged at 4 ° C, 5000 rpm for 10 minutes. The pellet is then taken up in a lysis buffer (10 mM Tris-HCl pH 7.5; 10 mM NaCl, 2 mM MgCl2). The cells thus taken up are then lysed, either with triton X-100 1% v / v, or lysozyme at 1 mg / ml of
  • RECTIFIED SHEET (RULE 91) ISA / EP lysis and left on ice about 5 to 10 min.
  • the cells are then thermodenatured by an exposure of 20 min at 72 ° C. This largely destroys the cells of the mesophilic host without destroying the recombinant proteins.
  • the lysis product is then centrifuged for 20 min at 10 000 rpm at 4 ° C. The supernatant is recovered in order to test it in incorporation, in PCR or on gel.
  • the incorporations are carried out according to two techniques: - With tritiated thymidine as tracer.
  • the incorporation is carried out on activated calf thymus (SIGMA Aldrich, F) in the following reaction medium: Tris-HCl 50mM pH 8.8; DTT ImM; MgCl2 10 mM; KCl 10MM;
  • the active fractions thus recovered are dialyzed for 5 h against a buffer C: 10 mM Tris-Ci pH 7.5; 0.5 mM EGTA; 5 mM MgCl2; 10 mM -mercaptoethanol; 0.2% Triton x100; 10% glycerol; 50 mM NaCl.
  • the products resulting from dialysis are successively loaded onto an affinity column for the proteins binding to DNA (Pharmacia, Blue HiTrap) and eluted in NaCl gradient with the same buffers as above.
  • the column is adjusted to 0.2 ml / min, the loading buffer A is composed of KP04 pH7 20mM, EDTA 0, lmM, DTT ImM, Glycerol 5%, Triton X-100 0.1% and the gradient (between 0% and 50% deB) is produced by KCl present in buffer B at 2M.
  • the second dialysis is carried out with the following buffer: K2HP04 pH 7.5, 10 lMM, K2PO4 lOmM, KCl 25 mM, DTT ImM, Triton X-100 0.1%, Glycerol 10%, for 1 hour.
  • the solution is then loaded onto a MonoQ column at 0.5 ml / min with a NaCl gradient of 0 to 20% (buffers used for heparin).
  • the 3 '-5' exonuclease assays are quantified by the release of P labeled nucleotides.
  • a first step makes it possible to carry out the labeling: the DNA of is digested with HindIII and then, the Klenow fragment copies the DNA from the free 3 '-OH ends, in a medium containing, in addition to the buffer, l DNA and the enzyme, 3 P dATP and 32 P dTTP, the dGTP and dCTP being cold. After one hour at 37 ° C, the four cold dNTPs are added in excess for half a hour. Kleno and dNTPs are removed by phenol extraction and precipitated with ethanol.
  • the exonuclease tests are carried out in solutions containing the enzyme buffers, 0.02 mg / ml of labeled DNA, and incubated overnight at 72 ° C, 80 ° C and 95 ° C. Different buffers containing MgCl2 or MnS04 are tested. The same test is carried out with the Wind as a positive control. 101 of reaction solution are then deposited on DE81 paper (Watmann), dried and then counted before and after washing (3 times 5 min with a2HP04, 1 time with water and then with 95% ethanol) using the technique from Cerenkov.
  • the labeling reaction uses the polynucleotide kinase to label the 5' substrate.
  • the Southern blot hybridization revealed fragments of two types: a HindIII-HindIII fragment of 1.9 kb and an Xhol-Xhol fragment of 5 kb. These two fragments were revealed only with the probe
  • RECTIFIED SHEET (RULE 91) ISAEP digested with Xhol. About 400 recombinants (E. Coli SURE) were screened with the Clal-HindIII probe. Of these 400 colonies, two gave a positive signal during hybridization. (n ° 9.26 and 12.79). The two clones were cultured in LB-Amp medium and their restriction profiles were identical, with an insert of 1.8 kb. The subsequent sequencing of one of these clones (designated 557MACa) and the sequence comparison (Megalign, DNASTAR program) have shown that it corresponds to the promoter region and to the first 1404 base pairs of a DNA gene. polymerase belonging to the family B (2).
  • fragment obtained by digestion of PCR product This fragment was cloned as before. About 200 recombinant clones were screened and four of them gave a positive signal. The four clones have an identical profile after digestion with the restriction enzyme HindIII. One of them, 557MACc, was sequenced and the sequence comparison demonstrated that it was the end of the DNA polymerase gene previously identified.
  • Thermococcus fumicolans is a new species of Thermococcales described by Godfroy et al (8).
  • the second is inserted at the base n ° 3157 and ends at 4323. It is also distributed between the clone 557MACb and 557MACC.
  • the coding section of the DNA polymerase gene therefore consists of three disjoint parts.
  • the first part of the gene, carried by 557MACa comprises the zone coding for the 3'-5 'exonuclease, where, after translation, the motif FDIET is recognized.
  • the second part, carried by 557MACb and the third part carried by 557MACC include the preserved sites SLYPSI, and YG.DTD.
  • the two insertion sequences are located on conserved areas of DNA polymerase, the first at the DFR / SLYPSII site such as I-KOD-1 of Pyrococcus sp. KOD1 and the second at the D / TDG site as T-I-Tli-1. l i toral i s.
  • I-Tfu-1 and I-Tfu-2 seem to represent two new "alleles” of "homing" archaebacterial endonucleases of classes A and C.
  • I-Tfu-1 is the third known allele of intein inserting into the A site of Archaea DNA polymerase, while I-Tfu-2 is the second of its class.
  • rTful00-2 clone Two other clones, also tested, are weakly inducible and express very little DNA polymerase. The rTful-1 and rTful00-2 clones are then tested in a 50 ml Erlenmeyer flask under the conditions described above. Only the inducible rTfulOO-2 clone has a constant volume expression of 50 ml. The rest of the work was therefore carried out on this clone.
  • the culture of the clone Tful00-2 was carried out in a 16 liter fermenter, in the 2xYT medium supplemented with ampicillin and chloramphenicol.
  • the fermenter has been prepared and put in temperature at 30 ° C with the culture medium.
  • the preculture was returned to 30 ° C one hour before its transfer to the fermenter.
  • the final culture volume is 15 liters.
  • the pH of the culture was adjusted to 7 during the acidification phase and then left free during the alkaline phase.
  • the bacteria were removed after four hours of induction, when the pH was 8.3.
  • the culture was then centrifuged and the cells were divided into three batches. One of them, 20 g of paste, was taken up in 80 ml of lysis buffer for further processing indicated in the Materials and methods chapter.
  • Fractions 54 to 60 are loaded at a rate of 0.25 ml / min. The elution makes it possible to recover 65 fractions of 5 ml with peaks of activity for fractions 36 to 56.
  • the dosage of the activity shows a concentration of 3 to 5 units for fractions 36 to 40.
  • FIG. 4 shows the result on SDS-PAGE gel. Following these three columns, the purity of the polymerase is significantly improved. Nevertheless, traces of DNA from E. coli attached to the polymerase and demonstrated by PCR remain. Two additional columns will therefore be implemented. 30ml of active fractions obtained previously are loaded onto a phosphocellulose column (this should fix the DNA and the polymerase differently).
  • the activity of the polymerase is identified by its high 3 '-5' exonuclease activity at 72 ° C. on the DNA of Lambda digested with HindIII. The strongest activity is located between fractions 40 and 49 with a sharp peak at 46 and 47. On these fractions 40 to 49 the pBR322 DNA is intact after overnight at 37 ° C.
  • Figure 5 shows us the results on gel with activities
  • T buffer Tris HC1 pH 8.8: 600mM; KCl: 500mM; MgC12: 15mM; Tween 20: 0.1%
  • FIG. 8 presents the results obtained with a reaction volume of 50 ⁇ l for quantities of DNA polymerase from Thermococcus fumicolans of 2.7 units. As it stands, the best results with Tfu are obtained with buffer R.
  • thermostability measured according to the protocol described above (Materials and Methods), or better, the residual activity in incorporation at 72 ° C. after exposure of the enzyme to high temperatures for variable times, is given in table 5 below. below.
  • thermostability which is lower than that of polymerases from more hyperthermophilic organisms such as Pyrococcus, is nonetheless very much greater than that of polymerases from Thermus and in particular all Taq.
  • the thermostability of the purified recombinant enzyme, both for the polymerase domain and for the exonuclease, is in any case very much higher than all known requirements in PCR.
  • GAG TTC GCC GAG GGG CCT ATT CTT ATG ATA AGC TAT GCC
  • GAC GAG GAA 954 Glu Phe Ala Glu Gly Pro Ile Leu Met Ile Ser Tyr Ala Asp Glu Glu 155 160 165
  • GTT GCC AAA AAA GAG GTT TAT CTT AGG GTT AGG GAA ATT ATG GAT GGC 2298 Val Ala Lys Lys Glu Val Tyr Leu Arg Val Arg Glu Ile Met Asp Gly 620 625 630
  • NAME / KEY DNA polymerase of THERMOCOCCUS fumicolans Tfu (xi) DESCRIPTION OF THE SEQUENCES: SEQ ID NO: 2:
  • Trp Glu Leu Ile Gly Leu Leu Val Gly Asp Gly Asn Trp Gly Gly Gin 145 150 155 160

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Abstract

The invention concerns a purified thermostable DNA polymerase, thermostable archae bacteria DNA polymerase of the Thermococcus fumicolans species having a molecular weight of the order of 89000 daltons and its thermostable inteins.

Description

ADN POLYMERASE THERMOSTABLE ET INTEINES DE L'ESPECE THERMOCOCCUS FUMICOLANS THERMOSTABLE DNA POLYMERASE AND INTEINS OF THE THERMOCOCCUS FUMICOLANS SPECIES
La présente invention concerne une nouvelle ADN polymerase thermostable ainsi que ses deux intéines, provenant d'une archaebactérie de l'espèce Thermococcus fumicolans .The present invention relates to a new thermostable DNA polymerase and its two inteins, originating from an archaebacterium of the species Thermococcus fumicolans.
Les ADN polymérases sont des enzymes impliquées dans la réplication et la réparation de 1 'ADN dans toute cellule vivante. On connaît aujourd'hui de nombreuses ADN polymérases isolées de micro-organismes tel que E. coliDNA polymerases are enzymes involved in the replication and repair of DNA in any living cell. Many DNA polymerases isolated from microorganisms such as E. coli are known today.
(ADN polymerase I) ou du phage T4. Des ADN polymérases ont aussi été identifiées et purifiées et à partir de micro-organismes thermophiles comme Thermus aqua ticus (Taq polymerase, Chien, A. et a l . J. Bact. 1976,(DNA polymerase I) or phage T4. DNA polymerases have also been identified and purified and from thermophilic microorganisms such as Thermus aqua ticus (Taq polymerase, Chien, A. et a. J. Bact. 1976,
127:1550-1557 ; Kaladin et al . Biokhymiya 1980, 45:644-127: 1550-1557; Kaladin et al. Biokhymiya 1980, 45: 644-
651), Thermus thermophilus , ou encore des espèces du genre Bacillus (demande de brevet Européen publiée sous le No. 699 760), Thermococcus (demande de brevet Européen No. 455 430), Sulfolobus et Pyrococcus (demande de brevet651), Thermus thermophilus, or species of the genus Bacillus (European patent application published under No. 699 760), Thermococcus (European patent application No. 455 430), Sulfolobus and Pyrococcus (patent application
Européen publiée sous le No. 547 359) . Parmi ces ADN polymérases issues d'archaebactéries on peut citer laEuropean published under No. 547 359). Among these DNA polymerases originating from archaebacteria, mention may be made of
Pfu , isolées de Pyrococcus furiosus (18), la Vent™ polymerase de Thermococcus li toralis (10), la 9°N de Pyrococcus sp. 9 °N (15) et la DeepVent™ de Pyrococcus GB-Pfu, isolated from Pyrococcus furiosus (18), Vent ™ polymerase from Thermococcus li toralis (10), 9 ° N from Pyrococcus sp. 9 ° N (15) and the DeepVent ™ from Pyrococcus GB-
D, les deux premières provenant de souches du littoral (Baie de Naples) , les deux suivantes de souches sous- marines profondes .D, the first two from littoral strains (Bay of Naples), the next two from deep underwater strains.
Le mécanisme d'action des ADN polymérases est aujourd'hui relativement bien connu et consiste en une réplication de l'ADN à l'identique selon un mode semi- conservatif. Le brin recopié sert de matrice et les quatre nucléotides triphosphates sont le substrat de cette polymérisation. Les enzymes ayant une activité ADN polymerase sont aujourd'hui de plus en plus utilisées in vi tro afin de travailler en biologie moléculaire dans divers buts tels le clonage, la détection d'erreurs, leThe mechanism of action of DNA polymerases is relatively well known today and consists of replication of DNA identically according to a semi-conservative mode. The copied strand serves as a matrix and the four nucleotide triphosphates are the substrate for this polymerization. Enzymes with DNA polymerase activity are increasingly used today in vitro to work in molecular biology for various purposes such as cloning, error detection,
FEUILLE RECTIFIEE (REGLE 91) ISA/EP séquençage, le marquage, et de façon générale, l'amplification de séquences d'acides nucléiques.RECTIFIED SHEET (RULE 91) ISA / EP sequencing, labeling, and generally speaking, amplification of nucleic acid sequences.
Cette amplification, in vi tro , de séquences d'acide désoxyribonucléique fait appel à la technique de la réaction de polymérisation en chaine (PCR) décrite dans les brevets Européens No. 200 362 et 201 184. Le principe de cette technique est basé sur la réalisation de cycles successifs d'extension d'amorces mettant en oeuvre les quatre nucléotides triphosphates ainsi qu ' une ADN polymerase et un ADN matrice à recopier. A chaque cycle, l'enzyme double le nombre de brins d'ADN disponibles et entre chaque cycle une thermodénaturation est nécessaire afin d'ouvrir la double hélice d'ADN pour le cycle suivant. Les températures utilisées pour cette étape de thermodénaturation ne sont pas compatibles avec la conservation de l'activité de la plupart des ADN polymérases connues, telle la Klenow. C'est ainsi que des nombreux efforts de recherche ont été réalisés afin de trouver des enzymes supportant ces températures . Cependant, si les micro-organismes thermophiles sont aujourd'hui connus, il reste encore difficile d'obtenir ces enzymes thermostables avec des rendements de production suffisants. La biologie moléculaire et le génie génétique permettent de palier cet inconvénient. Ainsi, une fois repéré dans le génome, le gène codant pour l'ADN polymerase est clone, séquence puis recloné dans un vecteur d'expression afin de produire la protéine dite alors recombinante, chez un hôte mésophile plus aisé à cultiver tel E. coli ou S. cerevisiae. Cette méthode d'expression chez E. coli a notamment été décrite dans la demande de brevet internationale PCT publiée sous le No. W0 89/06691 pour produire l'ADN polymerase de Thermus aquaticus .This amplification, in vi tro, of deoxyribonucleic acid sequences calls upon the technique of the polymerase chain reaction (PCR) described in European patents Nos. 200 362 and 201 184. The principle of this technique is based on the carrying out successive cycles of extension of primers using the four nucleotide triphosphates as well as a DNA polymerase and a template DNA to be copied. At each cycle, the enzyme doubles the number of DNA strands available and between each cycle thermodenaturation is necessary in order to open the DNA double helix for the next cycle. The temperatures used for this thermodenaturation step are not compatible with the conservation of the activity of most of the known DNA polymerases, such as Klenow. Many research efforts have been made to find enzymes that can withstand these temperatures. However, if thermophilic microorganisms are known today, it still remains difficult to obtain these thermostable enzymes with sufficient production yields. Molecular biology and genetic engineering overcome this drawback. Thus, once located in the genome, the gene coding for DNA polymerase is cloned, sequenced and then recloned in an expression vector in order to produce the so-called recombinant protein, in a mesophilic host which is easier to cultivate such as E. coli or S. cerevisiae. This expression method in E. coli has in particular been described in the international patent application PCT published under No. WO 89/06691 for producing DNA polymerase from Thermus aquaticus.
L'ADN polymerase de l'invention provient d'une archaebactérie de l'espèce Thermococcus fumicolans . Outre ses propriétés thermostables la rendant particulièrement efficace notamment dans une processus de PCR, cette ADNThe DNA polymerase of the invention comes from an archaebacterium of the species Thermococcus fumicolans. In addition to its thermostable properties making it particularly effective in particular in a PCR process, this DNA
FEUILLE RECTIFIEE (REGLE 91) ISAEP polymerase est remarquable en ce qu'elle présente deux " introns protéiques", encore appelés "intéines", au niveau de son polypeptide précurseur.RECTIFIED SHEET (RULE 91) ISAEP polymerase is remarkable in that it has two "protein introns", also called "inteins", at the level of its precursor polypeptide.
La séquence nucléotidique de ses intéines est insérée dans celle de l'ADN polymerase, généralement au niveau de sites conservés impliqués, après traduction, dans les réactions catalytiques . Ces séquences sont transcrites et traduites en même temps que celle de l'ADN polymerase et l'épissage autocatalytique des intéines produit alors trois enzymes: deux intéines et une ADN polymerase .The nucleotide sequence of its inteins is inserted into that of DNA polymerase, generally at the level of conserved sites involved, after translation, in catalytic reactions. These sequences are transcribed and translated at the same time as that of DNA polymerase and the autocatalytic splicing of the inteins then produces three enzymes: two inteins and one DNA polymerase.
On trouve de telles intéines également au sein d'autres molécules telles l'ATPase vacuolaire chez S . cerevisiae (4), GyrA chez Mycobacterium leprae (7), Rec A chez Mycobacterium tuberculosis (5, 6). Les intéines font partie pour leur majorité de la famille des endonucléases de type "homing endonucléases" puisqu'elles coupent l'ADN en un site reconnu, à l'endroit même où leur séquence nucléotidique vient s'insérer. Le développement des biotechnologies tant dans la recherche que dans les domaines de la médecine ou de 1 ' agro-alimentaire, nécessite de disposer de divers types d'ADN polymérases susceptibles d'améliorer quantitativement et qualitativement des techniques aussi diverses que le clonage, la détection, l'amplification de séquences d'ADN. La présente invention vise précisément à offrir une nouvelle ADN polymerase thermostable qui est issue d'une espèce récemment décrite: Thermococcus fumi colans (8) . Cet isolât a été isolé à partir de fragments de cheminées prélevées dans le bassin Nord- Fidgien lors de la campagne franco-japonnaise STARMER en 1989. Cette espèce, anaérobie stricte, présente une température optimale de croissance de 90°C, ce qui est relativement élevé pour un Thermococcus . Son pH optimum est de 8,8, et son taux de salinité de 20 g/1 à 40 g/1.Such inteins are also found in other molecules such as vacuolar ATPase in S. cerevisiae (4), GyrA in Mycobacterium leprae (7), Rec A in Mycobacterium tuberculosis (5, 6). The inteins belong for the most part to the family of endonucleases of the "homing endonucleases" type since they cut DNA at a recognized site, at the very place where their nucleotide sequence is inserted. The development of biotechnologies both in research and in the fields of medicine or agri-food, requires having various types of DNA polymerases capable of improving quantitatively and qualitatively techniques as diverse as cloning, detection , amplification of DNA sequences. The present invention aims precisely to offer a new thermostable DNA polymerase which is derived from a recently described species: Thermococcus fumi colans (8). This isolate was isolated from fragments of chimneys taken in the North Fidgian basin during the Franco-Japanese STARMER campaign in 1989. This species, strict anaerobic, has an optimal growth temperature of 90 ° C, which is relatively high for a Thermococcus. Its optimum pH is 8.8, and its salinity level from 20 g / 1 to 40 g / 1.
L'invention a donc pour objet une ADN polymerase purifiée thermostable d'archaebactéries deThe subject of the invention is therefore a purified thermostable DNA polymerase of archaebacteria of
FEUILLE RECTIFIEE (REGLE 91) ISA/EP l'espèce Thermococcus fumi colans ayant un poids moléculaire de l'ordre de 89000 Da ainsi que ses intéines thermostables , dont le gène comportant les deux séquences codant pour lesdites intéines a été clone.RECTIFIED SHEET (RULE 91) ISA / EP the species Thermococcus fumi colans having a molecular weight of the order of 89,000 Da as well as its thermostable inteins, the gene of which comprising the two sequences coding for said inteins has been cloned.
Les travaux de recherche ayant permis d'identifier, de sequencer et d'étudier l'ADN polymerase de 1 ' invention ont été réalisée à partir de la souche Thermococcus fumicolans ST557 déposée à la Collection de l'Institut Pasteur (CIP) sous le numéro CIP 104680. Cette ADN polymerase sera dénommée dans ce qui suit Tfu . Sa séquence de 774 acides aminés est représentée dans la liste de séquences en annexe sous le numéro SEQ ID NO: 2. Un poids moléculaire de 89797 Da et un pi de 8.1 ont été déduits de cette séquence.The research work which made it possible to identify, sequence and study the DNA polymerase of the invention was carried out using the strain Thermococcus fumicolans ST557 deposited in the Collection of the Institut Pasteur (CIP) under the number CIP 104680. This DNA polymerase will be named in the following Tfu. Its sequence of 774 amino acids is represented in the sequence list in the appendix under the number SEQ ID NO: 2. A molecular weight of 89797 Da and a pI of 8.1 have been deduced from this sequence.
L'invention concerne donc l'ADN polymerase purifiée thermostable d'archaebactéries de l'espèce Thermococcus fumicolans ayant un poids moléculaire de l'ordre de 89 000 daltons ainsi que ses dérivés enzymatiquement équivalents . On entend par dérivés enzymatiquement équivalent, les polypeptides et protéines constitués par ou comprenant la séquence en acides aminés représentée dans la liste de séquences en annexe sous le numéro SEQ ID NO : 2 dès lors qu'ils présentent les propriétés de l'ADN polymerase de Thermococcus fumi col ans . A ce titre l'invention envisage plus particulièrement une ADN polymerase dont la séquence en acides aminés est représentée dans la liste de séquence en annexe sous le numéro SEQ ID NO:l ou un fragment de celle-ci ou encore un assemblage de tels fragments, comme la séquence de 774 acides aminés représentée dans la liste de séquences en annexe sous le numéro SEQ ID NO: 2.The invention therefore relates to purified thermostable DNA polymerase of archaebacteria of the species Thermococcus fumicolans having a molecular weight of the order of 89,000 daltons as well as its enzymatically equivalent derivatives. The term “enzymatically equivalent derivatives” is understood to mean the polypeptides and proteins constituted by or comprising the amino acid sequence represented in the sequence list in the appendix under the number SEQ ID NO: 2 as soon as they exhibit the properties of the DNA polymerase of Thermococcus fumi col ans. As such, the invention more particularly contemplates a DNA polymerase, the amino acid sequence of which is represented in the sequence list in the appendix under the number SEQ ID NO: 1 or a fragment thereof or an assembly of such fragments, such as the 774 amino acid sequence represented in the sequence list in the appendix under the number SEQ ID NO: 2.
En effet, la présence de deux intéines I-Tfu-1 et I-Tfu-2 dans la séquence numéro SEQ ID NO:l, sont susceptibles de conduire lors de la préparation par synthèse chimique ou par génie génétique, à des séquences d'ADN polymerase de T. fumiculans tronquées dont lesIndeed, the presence of two inteins I-Tfu-1 and I-Tfu-2 in the sequence number SEQ ID NO: 1, are likely to lead during the preparation by chemical synthesis or by genetic engineering, to sequences of Truncated T. fumiculans DNA polymerase, the
FEUILLE RECTIFIEE (REGLE 91) ISA/EP propriétés enzymatiques sont équivalente à celle de l'ADN polymerase de T. fumicolans purifiée.RECTIFIED SHEET (RULE 91) ISA / EP Enzymatic properties are equivalent to that of purified T. fumicolans DNA polymerase.
On entend aussi par dérivés, les séquences en acides aminés ci-dessus modifiées par insertion et/ou délétion et/ou substitution d'un ou plusieurs aminoacides , pour autant que les propriétés de l'ADN polymerase de T. fumicolans qui en résultent ne soient pas significativement modifiées.The term “derivatives” is also understood to mean the amino acid sequences modified above by insertion and / or deletion and / or substitution of one or more amino acids, provided that the properties of the DNA polymerase of T. fumicolans which result therefrom are not significantly changed.
L'invention concerne également une séquence d'ADN constituée par ou comprenant la séquence codant pour une ADN polymerase de l'invention.The invention also relates to a DNA sequence consisting of or comprising the sequence coding for a DNA polymerase of the invention.
La séquence d'ADN représenté dans la liste de séquences en annexe sous le numéro SED ID NO: 1 représente une telle séquence. L'ADN codant pour l'ADN polymerase de T. fumicolans et ses deux intéines est constituée par le nucleotides 357 à 5028. Les nucleotides 1 à 356 correspondent à la région promotrice de ce gène. En conséquence, l'invention a pour objet une séquence d'ADN constituée par ou comprenant la séquence comprise entre les nucleotides 357 à 5028 de la SED ID NO: 1, ou un fragment de celle-ci ou encore un assemblage de tels fragmen s .The DNA sequence represented in the annexed sequence list under the number SED ID NO: 1 represents such a sequence. The DNA coding for the DNA polymerase of T. fumicolans and its two inteins consists of nucleotides 357 to 5028. Nucleotides 1 to 356 correspond to the promoter region of this gene. Consequently, the subject of the invention is a DNA sequence constituted by or comprising the sequence between nucleotides 357 to 5028 of SED ID NO: 1, or a fragment thereof, or an assembly of such fragmen s .
1 ' invention se rapporte tout particulièrement à une séquence d'ADN constituée par ou comprenant les nucleotides 357 à 1674 et 2755 à 3156 et 4324 à 5028 de la séquence d'ADN représenté dans la liste de séquences en annexe sous le numéro SED ID NO : 1.The invention relates more particularly to a DNA sequence constituted by or comprising nucleotides 357 to 1674 and 2755 to 3156 and 4324 to 5028 of the DNA sequence represented in the sequence list in the appendix under the number SED ID NO : 1.
Cette séquence code pour l'ADN polymerase de T. fumicolans dont la séquence de 774 acides aminés est représentée dans la liste de séquences en annexe sous le numéro SED ID NO : 2.This sequence codes for the DNA polymerase of T. fumicolans whose sequence of 774 amino acids is represented in the sequence list in the appendix under the number SED ID NO: 2.
L'invention concerne autant l'ADN polymerase isolées et purifiées de la souche Thermococcus fumicolans que l'ADN polymerase préparées par synthèse chimique, par exemple par ligature de fragments polypeptidiques , ou encore par les méthodes du génie génétique. Dans le cadre de ces méthodes du génie génétique, l'invention a aussi pour objet un vecteur comprenant une séquence d'ADN définie précédemment, ainsi qu'un procédé de production ou d'expression dans un hôte cellulaire des ADN thermostables de l'invention.The invention relates as much to DNA polymerase isolated and purified from the Thermococcus fumicolans strain as to DNA polymerase prepared by chemical synthesis, for example by ligation of polypeptide fragments, or also by genetic engineering methods. Within the framework of these genetic engineering methods, the invention also relates to a vector comprising a DNA sequence defined above, as well as a process for the production or expression in a cellular host of the thermostable DNAs of the invention. .
Un procédé de production d'une ADN polymerase conforme à l'invention consiste:A process for the production of a DNA polymerase according to the invention consists in:
- à transférer une séquence d'acide nucléique codant pour l'ADN polymerase ou un vecteur contenant ladite séquence dans un hôte cellulaire,transferring a nucleic acid sequence coding for DNA polymerase or a vector containing said sequence into a cell host,
- à cultiver l'hôte cellulaire obtenu à l'étape précédente dans des conditions permettant la production de l'ADN polymerase,to cultivate the cell host obtained in the previous step under conditions allowing the production of DNA polymerase,
- à isoler, par tous moyens appropriés, ladite ADN polymerase.- to isolate, by any appropriate means, said DNA polymerase.
L'hôte cellulaire mis en oeuvre dans les procédés précédents peut être choisi parmi les procaryotes ou les eucaryotes et notamment parmi les bactéries, les levures, les cellules de mammifères, de plantes ou d'insectes.The cell host used in the above methods can be chosen from prokaryotes or eukaryotes and in particular from bacteria, yeasts, mammalian, plant or insect cells.
Le vecteur utilisé est choisi en fonction de l'hôte dans lequel il sera transféré.The vector used is chosen according to the host to which it will be transferred.
L'ADN polymerase thermostable de l'invention est utile notamment dans les procédés d'amplification enzymatique de séquences d'acides nucléiques. En conséquence, l'invention a pour objet, de tels procédés mettant en oeuvre l'ADN polymerase thermostable décrite précédemment, ainsi que les kits d'amplification comprenant, outre les réactifs généralement utilisés, une quantité adéquate de cette ADN polymerase.The thermostable DNA polymerase of the invention is useful in particular in the methods of enzymatic amplification of nucleic acid sequences. Consequently, the subject of the invention is such methods using the thermostable DNA polymerase described above, as well as the amplification kits comprising, in addition to the reagents generally used, an adequate quantity of this DNA polymerase.
L'invention a également pour objet une intéine purifiée thermostable d'archaebactéries de l'espèce Thermococcus fumicolans. Comme indiqué précédemment, les intéines sont aussi définies comme des introns protéiques qui sont non pas épissés au niveau de l'ARN messager mais au niveauThe invention also relates to a purified thermostable intein of archaebacteria of the species Thermococcus fumicolans. As indicated previously, the inteins are also defined as protein introns which are not spliced at the level of the messenger RNA but at the level
FEUILLE RECTIFIEE (REGLE 91) ISA/EP d'une maturation proteique. Elles relèvent donc d'un seul gène traduit et transcrit en une seule étape, et constituent des sous produits de la maturation de la protéine codée par ce gène (Xu, M. G. , Comb, D.G ., Paulus H., Noren C.J., Shao Y., Perler, F., 1994, Protein splicing : an analysis of the branched intermediate and its resolution by succinimidine formation . EMBO J . 13, 5517-5522.)RECTIFIED SHEET (RULE 91) ISA / EP protein maturation. They therefore relate to a single gene translated and transcribed in a single step, and constitute by-products of the maturation of the protein encoded by this gene (Xu, MG, Comb, DG., Paulus H., Noren CJ, Shao Y ., Perler, F., 1994, Protein splicing: an analysis of the branched intermediate and its resolution by succinimidine formation. EMBO J. 13, 5517-5522.)
Les intéines sont des endonucléases de restriction qui ont la propriété de couper l'ADN à l'endroit même où s'insère leur gène, et en conséquence, elles peuvent être considérées comme des séquences égoïstes .The inteins are restriction endonucleases which have the property of cutting DNA at the very place where their gene is inserted, and therefore they can be considered as selfish sequences.
Les intéines possèdent dans leur séquence toute l'information nécessaire à leur propre épissage puisqu'elles s'épissent dans E. coli .The inteins have in their sequence all the information necessary for their own splicing since they splice in E. coli.
Il est possible de distinguer quatre grandes étapes de maturation proteique :It is possible to distinguish four main stages of protein maturation:
Une première étape de formation d'un intermédiaire linéaire qui possède une fonction ester.A first step in the formation of a linear intermediary which has an ester function.
Cette réaction est dépendante du pH et de l'environneme t local de cette liaison (nature des acides aminés) . Ce principe est utilisé dans les kits de clonage, d'expression ou de purification mettant en oeuvre des intéines, car un changement de l'environnement provoque ou non un épisssage. En effet, celui-ci serait inhibé à pH 11 et activé à pH 7,5.This reaction is dependent on the pH and the local environment of this bond (nature of the amino acids). This principle is used in cloning, expression or purification kits using inteins, because a change in the environment causes splicing or not. Indeed, it would be inhibited at pH 11 and activated at pH 7.5.
- Une deuxième étape de transestérif ication qui permet de transformer l'intermédiaire précédent et de déplacer l'équilibre de la première étape.- A second transesterification step which allows the previous intermediary to be transformed and to shift the balance of the first step.
La troisième étape consiste en une cyclisation de 1 ' asparagine libérant l'intéine.The third step consists in a cyclization of 1 asparagine releasing the intein.
- La quatrième étape est la stabilisation de la protéine mature et la formation d'une réelle liaison peptidique.- The fourth step is the stabilization of the mature protein and the formation of a real peptide bond.
Il est donc possible de construire des mutants thermosensibles permettant de bloquer l' épissageIt is therefore possible to construct thermosensitive mutants making it possible to block splicing.
FEUILLE RECTIFIEE (REGLE 91) ISA/EP proteique aux températures d'expression (30°C) et de 1 ' induire par chauffage .RECTIFIED SHEET (RULE 91) ISA / EP protein at expression temperatures (30 ° C) and induce it by heating.
Cette possibilité de contrôle de l' épissage proteique par la température peut-être utilisée dans des vecteurs de clonage avec une séquence codant pour 1 ' intéine et des sites de clonage autour. Si la protéine à cloner et à exprimer est toxique pour l'hôte, on peut la cloner en deux fragments autour de la séquence d' intéine. Ainsi, globalement, le gène à cloner est complet mais il est interrompu par la séquence de l' intéine. Lors de l'expression, 1 ' intéine se retrouve dans la protéine exprimée, la rendant ainsi inactive. Il est alors possible par chauffage, à la fin de l'induction, de libérer 1 ' intéine par épissage autocatalytique et ainsi retrouver la protéine clonée active .This possibility of controlling protein splicing by temperature can be used in cloning vectors with a sequence coding for the intein and around cloning sites. If the protein to be cloned and expressed is toxic to the host, it can be cloned into two fragments around the intein sequence. Thus, overall, the gene to be cloned is complete but it is interrupted by the sequence of the intein. During expression, the intein is found in the expressed protein, thus making it inactive. It is then possible by heating, at the end of the induction, to release the intein by autocatalytic splicing and thus find the cloned active protein.
Les intéines permettent ainsi de réaliser des purifications et sont utilisés dans des kits selon le principe décrit ci-après. Certains résidus autour de site d' épissage de 1 ' intéine sont modifiés. L'expression de la protéine recombinante est réalisée à basse température pour bloquer un éventuel épissage trop précoce. En C- terminal de l' intéine est fixé un site ayant une forte affinité pour la chitine. Lors de l'induction, la protéine clonée est exprimée ainsi que 1 ' intéine et le site de fixation à la chitine. La purification est alors réalisée avec la chitine, sur des colonnes d'affinité, qui retiennent la chitine et aussi 1 ' intéine et la protéine clonée, le tout faisant partie de la même pré- protéine. On hydrolyse alors l'extrémité N-terminale de 1 ' intéine avec du DTT ou du β-mercaptoéthanol pour libérer la protéine clonée.The inteins thus make it possible to carry out purifications and are used in kits according to the principle described below. Certain residues around the intein splicing site are modified. The expression of the recombinant protein is carried out at low temperature to block possible splicing too early. At the C-terminal of the intein is fixed a site having a strong affinity for chitin. During induction, the cloned protein is expressed as well as the intein and the chitin binding site. The purification is then carried out with the chitin, on affinity columns, which retain the chitin and also the intein and the cloned protein, the whole being part of the same pre-protein. The N-terminal end of the intein is then hydrolyzed with DTT or β-mercaptoethanol to release the cloned protein.
Les intéines sont aussi des endonucléases de restrictions thermostables, qui ont pour site de reconnaissance l'endroit même où s'insère leur gène dans la séquence "hôte" . Elles possèdent une séquence nucléique répétée deux fois (LAGLIDADG) dans la protéine,The inteins are also thermostable restriction endonucleases, which have as a recognition site the very place where their gene is inserted in the "host" sequence. They have a twice repeated nucleic sequence (LAGLIDADG) in the protein,
FEUILLE RECTIFIEE (REGLE 91) ISA/EP séquence plus ou moins conservée et qui correspond au site actif de reconnaissance et de coupure de l'ADN. Ces enzymes semblent aussi avoir besoin de g++ pour leur activité. II convient de remarquer que les deux intéines de 1 ' invention sont coexprimées dans E. coli et sont autoépissées . Ce qui signifie qu'elle n'ont pas de toxicité pour l'hôte, à la différence de l'une des intéines de Thermococcus li toralis (9), et en conséquence leur utilisation dans des kits d'expression ou de purification est aisée.RECTIFIED SHEET (RULE 91) ISA / EP more or less conserved sequence which corresponds to the active DNA recognition and cleavage site. These enzymes also seem to need g ++ for their activity. It should be noted that the two inteins of the invention are co-expressed in E. coli and are self-splicing. This means that they have no toxicity for the host, unlike one of the inteins of Thermococcus li toralis (9), and therefore their use in expression or purification kits is easy. .
Une première séquence d' intéine selon 1 ' invention est représentée dans la liste de séquences en annexe sous le numéro SEQ ID NO : 3. Cette intéine, dénommée I-Tfu-1, présente un poids moléculaire de 41 409 Da et un pi de 9,13, déduits de la séquence de 360 acides aminés de la séquence SEQ ID NO : 3.A first intein sequence according to the invention is represented in the annexed sequence list under the number SEQ ID NO: 3. This intein, called I-Tfu-1, has a molecular weight of 41,409 Da and a pi of 9.13, deduced from the 360 amino acid sequence of the sequence SEQ ID NO: 3.
Une seconde séquence d' intéine selon 1 ' invention est représentée dans la liste de séquences en annexe sous le numéro SEQ ID NO : 4. Cette intéine, dénommée I-Tfu-2, présente un poids moléculaire de 44 765 Da et pi de 9,6, déduits de la séquence de 389 acides aminés de la séquence SEQ ID NO: 4.A second intein sequence according to the invention is represented in the annexed sequence list under the number SEQ ID NO: 4. This intein, called I-Tfu-2, has a molecular weight of 44,765 Da and pi of 9 , 6, deduced from the 389 amino acid sequence of the sequence SEQ ID NO: 4.
Comme rappelé précédemment, les intéines thermostables de 1 ' invention sont utiles notamment dans les procédés de restriction d'acides nucléiques et dans la mise au point de vecteurs d'expression permettant de réduire la toxicité de la protéine à exprimer en insérant l'une des deux séquences d' intéines dans la séquence de la protéine à exprimer. Ceci peut se faire sans manipulation de la séquence des intéines si le clonage s'effectue chez E. coli , les techniques d'expression utilisées ayant démontré leur inocuité pour cet organisme hôte. En conséquence, l'invention a pour objet, de tels procédés mettant en oeuvre l'une des deux ou les deux intéines thermostables décrites précédemment, ainsi que les kits d'expression ou de purification contenant l'une ou les deux séquences codant pour les dites intéines thermostables .As recalled previously, the thermostable inteins of the invention are useful in particular in methods of restriction of nucleic acids and in the development of expression vectors making it possible to reduce the toxicity of the protein to be expressed by inserting one of the two sequences of inteins in the sequence of the protein to be expressed. This can be done without manipulation of the sequence of inteins if the cloning is carried out in E. coli, the expression techniques used having demonstrated their harmlessness for this host organism. Consequently, the subject of the invention is such processes using one or both of the two thermostable inteins described above, as well as the expression or purification kits containing one or the two sequences coding for said thermostable inteins.
L'invention concerne également une séquence d'ADN constituée par ou comprenant la séquence codant pour une intéine de 1 ' invention .The invention also relates to a DNA sequence consisting of or comprising the sequence coding for an intein of the invention.
Une séquence d'ADN codant pour 1 ' intéine I-Tfu-A DNA sequence coding for the I-Tfu intein
1 est comprise entre les nucleotides 1675 et 2754 dans la séquence SED ID NO: 1 en annexe. Cette séquence d'ADN code pour 1 ' intéine dont la séquence en acides aminés est représentée dans la liste de séquences en annexe sous le numéro SED ID NO : 3.1 is between nucleotides 1675 and 2754 in the sequence SED ID NO: 1 in the appendix. This DNA sequence codes for the intein, the amino acid sequence of which is represented in the annexed sequence list under the number SED ID NO: 3.
Une séquence d'ADN codant pour 1 ' intéine I-Tfu-A DNA sequence coding for the I-Tfu intein
2 est comprise entre les nucleotides 3157 et 4323 dans la séquence SED ID NO: 1 en annexe. Cette séquence d'ADN code pour l' intéine dont la séquence en acides aminés est représentée dans la liste de séquence en annexe sous le numéro SED ID NO : 4.2 is between nucleotides 3157 and 4323 in the sequence SED ID NO: 1 in the appendix. This DNA sequence codes for the intein whose amino acid sequence is represented in the sequence list in the appendix under the number SED ID NO: 4.
L'invention concerne autant ces intéines thermostables isolées et purifiées de la souche Thermococcus fumicolans que des intéines préparées par synthèse chimique, par exemple par ligature de fragments polypeptidiques, ou encore par les méthodes du génie génétique. Dans le cadre de ces méthodes du génie génétique, l'invention a aussi pour objet un vecteur comprenant une séquence d'ADN définie précédemment, ainsi qu'un procédé de production ou d'expression dans un hôte cellulaire des ADN codant pour les intéines de l'invention. De tels procédés sont identiques à ceux rapportés précédemment pour l'ADN polymerase de T . fumicolans .The invention relates both to these thermostable inteins isolated and purified from the Thermococcus fumicolans strain as to inteins prepared by chemical synthesis, for example by ligation of polypeptide fragments, or also by genetic engineering methods. Within the framework of these methods of genetic engineering, the invention also relates to a vector comprising a DNA sequence defined above, as well as a process for the production or expression in a cellular host of DNA coding for the inteins of the invention. Such methods are identical to those reported previously for T DNA polymerase. fumicolans.
D'autres avantages et caractéristiques de 1 ' invention apparaîtront à la lecture des exemples qui suivent, donnés à titre non limitatifs et concernant le clonage, l'expression, la caractérisation et l'activitéOther advantages and characteristics of the invention will become apparent on reading the examples which follow, given without limitation and relating to cloning, expression, characterization and activity
FEUILLE RECTIFIEE (REGLE 91) ISA/EP de l'ADN thermostable de l'invention, et se référant aux dessins annexés dans lesquels :RECTIFIED SHEET (RULE 91) ISA / EP of the thermostable DNA of the invention, and referring to the accompanying drawings in which:
- La figure 1 représente l'hybridation ADN-ADN de l'ADN genomique de T. fumicolans digéré par diverses enzymes de restriction et hybride avec les sondes GE23ClaI-HindIII et GE23XhoI-SalI .- Figure 1 shows the DNA-DNA hybridization of the genomic DNA of T. fumicolans digested with various restriction enzymes and hybridized with the probes GE23ClaI-HindIII and GE23XhoI-SalI.
- La figure 2 représente la stratégie de clonage, la structure du gène de l'ADN polymerase de T. fumiculans et les produits du gène. - La figure 3 représente les résultats de purification de la polymerase recombinante de T. fumi colans après une colonne d'héparine sépharose, visualisés par SDS-PAGE.- Figure 2 shows the cloning strategy, the gene structure of the DNA polymerase of T. fumiculans and the gene products. - Figure 3 shows the results of purification of the recombinant polymerase of T. fumi colans after a heparin sepharose column, visualized by SDS-PAGE.
- La figure 4 représente les résultats de purification de la polymerase recombinante de T. fumiculans après une colonne Bleue HTrap n°2 , visualisé par SDS-PAGE.- Figure 4 represents the results of purification of the recombinant polymerase of T. fumiculans after a Blue column HTrap n ° 2, visualized by SDS-PAGE.
- La figure 5 représente les résultats de purification de la polymerase recombinante de T. fumi col ans après une colonne de phosphocellulose , visualisés par SDS-PAGE.- Figure 5 shows the purification results of the recombinant polymerase of T. fumi col years after a phosphocellulose column, visualized by SDS-PAGE.
- La figure 6 représente les résultats de purification de la polymerase recombinante de T. fumicolans après une colonne MonoQ, visualisés par SDS- PAGE.- Figure 6 represents the results of purification of the recombinant polymerase of T. fumicolans after a MonoQ column, visualized by SDS-PAGE.
- La figure 7 représente les résultats de PCR avec l'ADN polymerase de T. fumicolans avec les fractions d'exclusion de la colonne MonoQ.- Figure 7 shows the PCR results with DNA polymerase from T. fumicolans with the exclusion fractions from the MonoQ column.
I- Matériel et méthodes .I- Materials and methods.
1) Conditions de culture, plasmides et souches utilisées . Les souches Thermococcus li toralis (DSM 5474 T) et Pyrococcus furiosus (DSM 3638 T ) ont été obtenues auprès de la collection du Deutsche Sammlung von1) Culture conditions, plasmids and strains used. The strains Thermococcus li toralis (DSM 5474 T) and Pyrococcus furiosus (DSM 3638 T) were obtained from the collection of the Deutsche Sammlung von
Microorganismen (DSM) Braunschweig-Stocheim, Allemagne.Microorganismen (DSM) Braunschweig-Stocheim, Germany.
FEUILLE RECTIFIEE (REGLE 91) ISAEP La souche Pyrococcus sp. GE 23 a été isolée de cheminées de sources hydrothermales profondes et a été fournie par G Erauso (CNRS, Station Biologique de Roscoff , France) . La souche Thermococcus fumicolans à été obtenue auprès du laboratoire de Microbiologie Marine de G. Barbier (IFREMER-DRV-VP-CMM) à Brest, France. Cette souche, Thermococcus fumicolans à été obtenue par purification à partir de fragments de cheminées hydrothermales receuillies dans le bassin nord-Fidgien lors de la campagne franco- aponaise STARMER effectuée en 1989 à 2000 mètres de profondeur.RECTIFIED SHEET (RULE 91) ISAEP The strain Pyrococcus sp. GE 23 was isolated from chimneys of deep hydrothermal vents and was supplied by G Erauso (CNRS, Station Biologique de Roscoff, France). The Thermococcus fumicolans strain was obtained from the Marine Microbiology laboratory of G. Barbier (IFREMER-DRV-VP-CMM) in Brest, France. This strain, Thermococcus fumicolans, was obtained by purification from fragments of hydrothermal vents collected in the North Fidgian basin during the Franco-Aponais campaign STARMER carried out in 1989 at 2000 meters depth.
Pyrococcus sp.GE23 a été cultivée à 85°C dans un milieu 2216S (DIFCO) à un pH de 6 , 5.Pyrococcus sp.GE23 was cultured at 85 ° C in 2216S medium (DIFCO) at a pH of 6.5.
Thermococcus fumi col ans , décrite en 1996 (Godfroy et al.) est cultivée en conditions anaérobies dans un milieu contenant les éléments suivants: peptone 2g/ 1; extraits de levure 0,5g/l; sel de mer (Sigma) 30g/l; tampon PIPES 6,05g/l, soufre élémentaire 10g/1, rézasurine lmg/1. Le pH est ajusté à 8,5 par de la soude 5N à 20°C.Thermococcus fumi col ans, described in 1996 (Godfroy et al.) Is cultivated under anaerobic conditions in a medium containing the following elements: peptone 2g / 1; yeast extracts 0.5 g / l; sea salt (Sigma) 30g / l; PIPES buffer 6.05g / l, elemental sulfur 10g / 1, rezasurin lmg / 1. The pH is adjusted to 8.5 with 5N sodium hydroxide at 20 ° C.
La souche de Escherichia coli SURE (Stratagene, La Jolla, Calif.) a été cultivée dans du milieu LB avec le ou les antibiotiques appropriés, à 37°C sous agitation. Cette souche a été utilisée comme hôte pour recevoir les constructions primaires à partir des vecteurs pUC 18 ou pBluescript. Les souches NovaBlue, BL21(DE3) et BL21(DE3)pLysS (Novagen, Madison, Wi . ) ont été cultivées dans du milieu 2xYT avec les antibiotiques appropriés à 37°C ou 30°C. Ces souches ont été utilisées commes hôtes pour les plasmides d'expression.The Escherichia coli SURE strain (Stratagene, La Jolla, Calif.) Was cultured in LB medium with the appropriate antibiotic (s), at 37 ° C. with stirring. This strain was used as a host to receive the primary constructs from the pUC 18 or pBluescript vectors. The NovaBlue, BL21 (DE3) and BL21 (DE3) pLysS strains (Novagen, Madison, Wi.) Were cultured in 2xYT medium with the appropriate antibiotics at 37 ° C or 30 ° C. These strains were used as hosts for the expression plasmids.
2) Isolement de l'ADN, hybridation et ADN recombinants .2) Isolation of DNA, hybridization and recombinant DNA.
L'ADN de haut poids moléculaire de Thermococcus fumicolans a été isolé par la méthode de Charbonnier modifiée (3) . Les cellules ont été resuspendues dans un tampon TE-Na IX, puis lysées à 40°C pendant trois heuresThe high molecular weight DNA of Thermococcus fumicolans was isolated by the modified Charbonnier method (3). The cells were resuspended in TE-Na IX buffer, then lysed at 40 ° C for three hours
FEUILLE RECTIFIEE (REGLE 91) ISA/EP avec un mélange de N-Lauryl-sarcosine 1%, dodecyl sulfate de sodium 1% et 0,4 mg/ml de protéinase K. Après la lyse, une centrifugation à 5000g pendant 10 minutes permet d'éliminer les débris cellulaires. L'ADN est extrait par un traitement au Phenol-Chloroforme-alcool Isoamylique ou PCI (25-24-1) , puis traité par la RNAse à 5μg/ml à 60°C pendant une heure. Ces étapes sont suivies de deux extractions additionelles par PCI et d'une extraction au chlorophorme . L'ADN est ensuite précipité à l'éthanol absolu à -20°C, puis centrifugé, séché à l'air et repris dans un tampon TE-lx. La concentration et la pureté de cet ADN sont mesurées par spectrophotométrie à 230, 260 et 280nm avec un appareil GeneQuantlI (Pharmacia, Upsalla, Sweden) . Pour la construction de la mini-banque genomique en pUC 18 (17), l'ADN a été totalement digéré pendant une nuit à 37°C par une série d'enzymes de restriction (BamHI, HindIII, EcoRI , EcoRV, PvuII, Sali, Xbal et Xhol) par simples et doubles digestions. Puis les fragments d'ADN sont fractionnés sur gel d'agarose à 0,8% dans de TBE-1X et transférés sous vide sur une membrane de nylon Hybond-N+ (Amersham, UK) . Ces membranes ont été hybridées avec des sondes d'ADN préparées par PCR avec des amorces spécifiques sélectionnées à partir des gènes d'ADN polymérases de P . furi osus , T . l i toral i s et Pyrococcus sp . GE23. Ces sondes sont préalablement marquées au 32P par "random primmg" conformément aux recommandations du fabricant (Megaprime, Amersham, UK) .RECTIFIED SHEET (RULE 91) ISA / EP with a mixture of 1% N-Lauryl-sarcosine, 1% sodium dodecyl sulfate and 0.4 mg / ml of proteinase K. After lysis, centrifugation at 5000 g for 10 minutes makes it possible to remove cellular debris. The DNA is extracted by a treatment with Phenol-Chloroform-Isoamyl alcohol or PCI (25-24-1), then treated with RNAse at 5 μg / ml at 60 ° C for one hour. These steps are followed by two additional PCI extractions and a chlorophoric extraction. The DNA is then precipitated with absolute ethanol at -20 ° C, then centrifuged, air dried and taken up in TE-1x buffer. The concentration and purity of this DNA are measured by spectrophotometry at 230, 260 and 280nm with a GeneQuantlI device (Pharmacia, Upsalla, Sweden). For the construction of the genomic mini-bank in pUC 18 (17), the DNA was completely digested overnight at 37 ° C. by a series of restriction enzymes (BamHI, HindIII, EcoRI, EcoRV, PvuII, Sali , Xbal and Xhol) by simple and double digestions. Then the DNA fragments are fractionated on 0.8% agarose gel in TBE-1X and transferred in vacuo to a Hybond-N + nylon membrane (Amersham, UK). These membranes were hybridized with DNA probes prepared by PCR with specific primers selected from the DNA polymerase genes of P. furi osus, T. li toral is and Pyrococcus sp. GE23. These probes are previously marked with 32P by "random primmg" in accordance with the manufacturer's recommendations (Megaprime, Amersham, UK).
Deux sondes de P. furiosus ont été utilisées, Pfu et PfuTwo P. furiosus probes were used, Pfu and Pfu
F, couvrant respectivement les régions délimitées par les paires de bases 8 à 2316 et 819 à 1915 de la section codante du gène de la polymerase comme défini par Uemori et al . (18) . Deux sondes de T. li toralis, Tli I et Tli T, couvrant respectivement les régions délimitées par les paires de bases 297 à 1768 et 4631 à 5378, comme défini par Hodges et al . (9) . Deux sondes de Pyrococcus sp GE23 ont été utilisées, l'une contenant la partie 5' du gène (fragment Clal-HindIIIF, covering respectively the regions delimited by base pairs 8 to 2316 and 819 to 1915 of the coding section of the polymerase gene as defined by Uemori et al. (18). Two T. li toralis probes, Tli I and Tli T, covering the regions delimited by base pairs 297 to 1768 and 4631 to 5378 respectively, as defined by Hodges et al. (9). Two Pyrococcus sp GE23 probes were used, one containing the 5 'part of the gene (Clal-HindIII fragment
FEUILLE RECTIFIEE (REGLE 91) ISA/EP correspondant aux sites pb 8 et pb 1353 de la section codante) et l'autre contenant la partie terminale de ce même gène obtenu par PCR (amorces dites d'expression Ndel et Sali correspondant aux sites pb -1 et pb 2318, puis digestion par Xhol et Sali comprenant les bases du n° 1879 à 2318) . Les fragments positifs ont été identifiés par hybridation ADN-ADN (14). Seules les hybridations avec les sondes de Pyrococcus sp . GE 23 ont fourni des signaux positifs à 55°C en moins de 24 h d'exposition. Les sondes issues de T. li toralis et de P. furiosus n'ont donné aucun résultat, même à 50°C dans un tampon standard sans formamide . A partir des hybridations avec les sondes de Pyrococcus sp. GE 23, des fragments HindIII de 1,9 kb ont été sélectionnés, puis préparés par une digestion appropriée de lOOμg d'ADN genomique, purifiés dans des sacs de dialyse à partir des gels d'agarose, et précipités à l'éthanol absolu après une extraction au PCI. Les fragments ont été ligaturés dans un pUC 18/HindIII déphosphorylé. Les transformations des souches hôtes ont été réalisées par électroporation (Gène Puiser, Biorad) . Le criblage des clones recombinants a été effectué par sélection à 1 ' ampicilline , alpha- complémentation sur substrat X-Gal-IPTG puis hybridation de colonies selon les techniques standards (12) . La température des hybridations de colonies était de 55°C avec les sondes de Pyrococcus sp GE23, dans un tampon standard sans formamide. L'ADN plasmidique a été isolé selon la méthode décrite par Birnboim et Doly (1) , puis purifié par chromatographie échangeuse d'anions en phase solide (Quiagen, Chatsworth, Calif.). Les fragments de restriction des plasmides ont été purifiés sur gel d'agarose par la méthode du GeneClean (Bio 101, La Jolla, Calif.) pour un clonage ultérieur.RECTIFIED SHEET (RULE 91) ISA / EP corresponding to sites pb 8 and pb 1353 of the coding section) and the other containing the terminal part of this same gene obtained by PCR (primers known as expression Ndel and Sali corresponding to sites pb -1 and pb 2318, then digestion by Xhol and Sali including the bases of Nos. 1879 to 2318). Positive fragments were identified by DNA-DNA hybridization (14). Only hybridizations with Pyrococcus sp. GE 23 provided positive signals at 55 ° C in less than 24 h of exposure. The probes from T. li toralis and P. furiosus gave no results, even at 50 ° C in a standard buffer without formamide. From hybridizations with the Pyrococcus sp. GE 23, HindIII fragments of 1.9 kb were selected, then prepared by appropriate digestion of 100 μg of genomic DNA, purified in dialysis bags from agarose gels, and precipitated with absolute ethanol after PCI extraction. The fragments were ligated into a dephosphorylated pUC 18 / HindIII. The transformations of the host strains were carried out by electroporation (Gene Puiser, Biorad). The screening of the recombinant clones was carried out by selection with ampicillin, alpha-complementation on X-Gal-IPTG substrate then hybridization of colonies according to standard techniques (12). The temperature of the colony hybridizations was 55 ° C. with the Pyrococcus sp GE23 probes, in a standard buffer without formamide. The plasmid DNA was isolated according to the method described by Birnboim and Doly (1), then purified by anion exchange chromatography in solid phase (Quiagen, Chatsworth, Calif.). The restriction fragments of the plasmids were purified on agarose gel by the GeneClean method (Bio 101, La Jolla, Calif.) For later cloning.
L'ARNr 16S et 23S de Thermococcus fumicolans a été amplifié par PCR en utilisant les amorces suivantes :Thermococcus fumicolans 16S and 23S rRNA was amplified by PCR using the following primers:
- amorce directe Aa: 5' TCCGGTTGATCCTGCCGGAA-3 '- direct primer Aa: 5 'TCCGGTTGATCCTGCCGGAA-3'
- amorce réverse 23Sa: 5 ' -CTTTCGGTCGCCCCTACT-3 '- 23Sa reverse primer: 5 '-CTTTCGGTCGCCCCTACT-3'
FEUILLE RECTIFIEE (REGLE 91) ISAEP - étape initiale 3 minutes à 94°C suivi de 30 cycles (94°C, 1min/ 49°C, lmn/ 72°C, 2mn) et, élongation finale de 5 irai à 72°C.RECTIFIED SHEET (RULE 91) ISAEP - initial step 3 minutes at 94 ° C followed by 30 cycles (94 ° C, 1min / 49 ° C, lmn / 72 ° C, 2mn) and, final elongation of 5 will go to 72 ° C.
Le produit de PCR a été clone dans le vecteur pUC18 pour séquençage ultérieur.The PCR product was cloned into the vector pUC18 for subsequent sequencing.
3 ) Sé uencaσe des ADN.3) Se uencaσe of DNA.
Les séquences d'ADN ont été obtenues par la méthode de terminaison de chaine (13) en utilisant un système d'analyses automatique d'ADN Applied Biosyste s. Les deux brins des gènes codant pour l'ADN polymerase et les deux intéines ont été séquences en utilisant des amorces universelles localisées sur des vecteurs ainsi que des amorces internes . La séquence d'ADNr 16S a été réalisée sur les deux brins, après clonage (SureClone, Pharmacia, Uppsala, Sweeden) , en utilisant le kit Hot-Tub (Amersham, UK. ) afin de lever les compressions .The DNA sequences were obtained by the chain termination method (13) using an Applied Biosyste s automatic DNA analysis system. The two strands of the genes coding for DNA polymerase and the two inteins were sequenced using universal primers localized on vectors as well as internal primers. The 16S rDNA sequence was carried out on both strands, after cloning (SureClone, Pharmacia, Uppsala, Sweeden), using the Hot-Tub kit (Amersham, UK.) In order to remove the compressions.
L'analyse de séquence a été réalisée avec le logiciel DNASTAR (Madison, Wis . , USA) et le programme de Genetic Computer Group (University of Wisconsin Biotechnology Center, Wis., USA) accessible en ligne sur INFOBIOGEN. Les recherches informatisées de similitude ont été réalisées avec le programme BLAST, les alignements multiples avec CLUSTAL V, et les arbres phylogénétiques ont été établis selon la méthode dite de "Neighbour- oining" (11).Sequence analysis was performed with DNASTAR software (Madison, Wis., USA) and the program of Genetic Computer Group (University of Wisconsin Biotechnology Center, Wis., USA) available online on INFOBIOGEN. Computerized similarity searches were performed with the BLAST program, multiple alignments with CLUSTAL V, and phylogenetic trees were established using the so-called Neighbouring method (11).
4 ) Construction du recombinant exprimant l'ADN polymerase de Thermococcus fumicolans .4) Construction of the recombinant expressing the DNA polymerase of Thermococcus fumicolans.
L'ADN polymerase de Thermococcus fumicolans ainsi que ses deux intéines, ont été exprimées en même temps chez E. coli avec le vecteur d'expression PARHS2 qui appartient à la famille des systèmes d'expression T7 ( 16 ) acquis auprès d ' Eurogentec .The DNA polymerase of Thermococcus fumicolans as well as its two inteins, were expressed at the same time in E. coli with the expression vector PARHS2 which belongs to the family of expression systems T7 (16) acquired from Eurogentec.
La PCR a été utilisée pour préparer le fragment complet de l'ADN polymerase et des deux intéines en utilisant des amorces contenant les sites de restriction Ndel et BamHI :PCR was used to prepare the complete fragment of DNA polymerase and the two inteins in using primers containing the NdeI and BamHI restriction sites:
- amorce Tfu Dir : 5 ' -TGG GGA TCC ATA TGA TCC TCG ATA CAG ACT ACA TC-3 ' - amorce Tfu Rev :- primer Tfu Dir: 5 '- TGG GGA TCC ATA TGA TCC TCG ATA CAG ACT ACA TC-3' - primer Tfu Rev:
5 ' -AAG CTT GGA TCC TCA TTT CTT CCC CAT TTT GAG CC-3 '5 '-AAG CTT GGA TCC TCA TTT CTT CCC CAT TTT GAG CC-3'
Le mélange réactionnel contenait l'ADN polymerase GOLDSTAR (Eurogentec, B) , l'enzyme Taq Extender (contenant la Pfu de Stratagene) , le tampon d'extension avec les quatre dNTP (chacun à 0,2mM) et les amorces Tfu Dir et Tfu Rev à 50 pmoles dans un volume de 50μl final. L'amplification a été effectuée sur 20 cycles : 1 mn 94°C, 1 mn à 54°C et 6 mn à 72°C en utilisant un thermocycleur Stratagene 96-gradient. Les fragments de PCR ont ensuite été digérés par les enzymes Ndel et BamHI puis ligaturés aux mêmes sites du vecteur, rétablissant ainsi le codon d'initiation. La construction ainsi obtenue a été nommée PARHS2TFU1. Cette construction a été séquencée aux sites de jonction afin de vérifier son intégrité par rapport à la séquence de l'ADN genomique. Les tests d'expression ont été réalisés selon le protocole suivant: sélection des clones recombinants dans la souche E. coli Novablue, expression avec la souche BL21 (DE3 )pLysS dans un milieu 2xYT et induction de quatre heures à ImM d'IPTG.The reaction mixture contained GOLDSTAR DNA polymerase (Eurogentec, B), the Taq Extender enzyme (containing Pfag from Stratagene), the extension buffer with the four dNTPs (each at 0.2mM) and the primers Tfu Dir and Tfu Rev at 50 pmol in a volume of 50 μl final. The amplification was carried out over 20 cycles: 1 min 94 ° C, 1 min at 54 ° C and 6 min at 72 ° C using a Stratagene 96-gradient thermocycler. The PCR fragments were then digested with the enzymes NdeI and BamHI and then ligated to the same sites of the vector, thus restoring the initiation codon. The construction thus obtained was named PARHS2TFU1. This construction was sequenced at the junction sites to verify its integrity with respect to the genomic DNA sequence. The expression tests were carried out according to the following protocol: selection of the recombinant clones in the E. coli Novablue strain, expression with the BL21 (DE3) pLysS strain in a 2xYT medium and induction for four hours at ImM of IPTG.
Les premiers essais d'induction ont été réalisés sur des cultures de 5 ml, induites ou non. Des précultures de nuit sont réalisées sans inducteur et relancées dans un milieu frais le matin (au dizième) , jusqu'à ce que la densité optique (mesurée à 600nm) soit de 0,6, puis soit induites pendant 4 heures, soit non induites et arrêtées au bout de 2 , 4 ou 14 heures . Quatre ml de cultures sont alors centrifugés à 4°C, 5000rpm pendant 10 minutes. Le culot est ensuite repris dans un tampon de lyse (Tris-HCl lOmM pH 7,5; NaCl lOmM, MgCl2 2 mM) . Les cellules ainsi reprises sont alors lysées , soit avec du triton X-100 1% v/v, soit du lysozyme à lmg/ml deThe first induction tests were carried out on 5 ml cultures, whether induced or not. Night precultures are carried out without an inductor and restarted in a fresh medium in the morning (to the tenth), until the optical density (measured at 600nm) is 0.6, then either induced for 4 hours, or not induced and stopped after 2, 4 or 14 hours. Four ml of cultures are then centrifuged at 4 ° C, 5000 rpm for 10 minutes. The pellet is then taken up in a lysis buffer (10 mM Tris-HCl pH 7.5; 10 mM NaCl, 2 mM MgCl2). The cells thus taken up are then lysed, either with triton X-100 1% v / v, or lysozyme at 1 mg / ml of
FEUILLE RECTIFIEE (REGLE 91) ISA/EP lyse et laissées sur glace environ 5 à 10 min. Les cellules sont ensuite thermodénaturées par une exposition de 20 min à 72°C. Ceci permet de détruire en grande partie les cellules de l'hôte mésophile sans détruire les protéines recombinantes . Le produit de lyse est ensuite centrifugé 20 min à 10 OOOrpm à 4°C. Le surnageant est récupéré afin de le tester en incorporation, en PCR ou sur gel. Les incorporations sont réalisées suivant deux techniques : - Avec de la thymidine tritiée comme traceur.RECTIFIED SHEET (RULE 91) ISA / EP lysis and left on ice about 5 to 10 min. The cells are then thermodenatured by an exposure of 20 min at 72 ° C. This largely destroys the cells of the mesophilic host without destroying the recombinant proteins. The lysis product is then centrifuged for 20 min at 10 000 rpm at 4 ° C. The supernatant is recovered in order to test it in incorporation, in PCR or on gel. The incorporations are carried out according to two techniques: - With tritiated thymidine as tracer.
L'incorporation est réalisée sur du thymus de veau activé (SIGMA Aldrich, F) dans le milieu de réaction suivant: Tris-HCl 50mM pH 8,8 ; DTT ImM ; MgCl2 lOmM ; KCl lOmM ;The incorporation is carried out on activated calf thymus (SIGMA Aldrich, F) in the following reaction medium: Tris-HCl 50mM pH 8.8; DTT ImM; MgCl2 10 mM; KCl 10MM;
BSA 0,4 mg/ml; chaque dNTP à 0,4 mM. - Avec du 32 P -dATP (Amersham) comme traceur.BSA 0.4 mg / ml; each dNTP at 0.4 mM. - With 32 P -dATP (Amersham) as a tracer.
L'incorporation est réalisée sur de l'ADN thymus de veau activé (Appligene) dans le milieu de réaction suivant:The incorporation is carried out on activated calf thymus DNA (Appligene) in the following reaction medium:
Tris-HCl pH 9 50mM; KCl 50mM; MgCl2 7mM; BSA 0,2mg/ml et50 mM Tris-HCl pH 9; 50 mM KCl; 7mM MgCl2; BSA 0.2mg / ml and
(NH4+)S04 (filtré) 16mM, avec un mélange des 4 dNTP à 500μM final chacun. Cette seconde méthode permet d'estimer avec précision le nombre d'unités d'enzyme.(NH4 +) S04 (filtered) 16mM, with a mixture of the 4 dNTPs at 500μM final each. This second method makes it possible to accurately estimate the number of units of enzyme.
5) Purification.5) Purification.
a) Culture.a) Culture.
Après des essais en petits volumes, les cultures destinées à l'expression de l'enzyme recombinante ont été réalisées comme suit: production d'un inoculum de 700 ml (milieu 2x YT complémenté en ampicilline et chloramphenicol) cultivé à 30°C jusqu'à DO = 0,8; ensemencement d'un fermenteur contenant 16 1 du même milieu; culture pendant 4 h jusqu'à DO= 0,6, puis induction à l'iPTG 1 mM et culture pendant 4 h. La biomasse résultante est centrifugée 20 mn à 6 000 rpm à 4°C (Centrifugeuse JOUAN) .After tests in small volumes, the cultures intended for the expression of the recombinant enzyme were carried out as follows: production of an inoculum of 700 ml (2x YT medium supplemented with ampicillin and chloramphenicol) cultivated at 30 ° C. until at DO = 0.8; inoculation of a fermenter containing 16 1 of the same medium; culture for 4 h until OD = 0.6, then induction with 1 mM iPTG and culture for 4 h. The resulting biomass is centrifuged for 20 min at 6000 rpm at 4 ° C (JOUAN centrifuge).
FEUILLE RECTIFIEE (REGLE 91) ISAEP b) Lyse cellulaire et première étape de purification.RECTIFIED SHEET (RULE 91) ISAEP b) Cell lysis and first purification step.
20 g de biomasse sont repris dans 80 ml de tampon (20 mM Tris-Ci pH 7 , 5 ; 10 mM NaCl; 2 mM MgC12 ; 1 mM EGTA; 1% Triton xlOO; 2,2 mM PMSF) . Le mélange résultant, maintenu à 4°C maximum, est soniqué à 12 reprises successives (cycle de 15 s) jusqu'à obtention d'une solution liquide. Le surnageant est ensuite centrifugé 20 mn à 4°C à 20 000 rpm (SORVALL Ti45) . Le surnageant est récupéré et traité par la chaleur (70°C pendant 10 mn) afin de thermodénaturer l'essentiel des protéines natives de E. coli , puis centrifugé à nouveau.20 g of biomass are taken up in 80 ml of buffer (20 mM Tris-Ci pH 7.5, 10 mM NaCl; 2 mM MgC12; 1 mM EGTA; 1% Triton xlOO; 2.2 mM PMSF). The resulting mixture, kept at 4 ° C maximum, is sonicated 12 successive times (15 s cycle) until a liquid solution is obtained. The supernatant is then centrifuged for 20 min at 4 ° C at 20,000 rpm (SORVALL Ti45). The supernatant is recovered and treated with heat (70 ° C. for 10 min) in order to thermodenature most of the native proteins of E. coli, then centrifuged again.
c) Chromatoσraphie . Les 70 ml de surnageant issus de l'étape précédente sont chargés sur une colonne Pharmacia Héparine Sépharose (30 ml de résine) , après équilibration avec un tampon A (10 mM Tris-Ci pH 7 , 5 ; 0 , 5 mM EGTA; 5 mM MgCl2; 10 mM β-mercaptoéthanol ; 0,2% Triton xlOO et 10% glycerol. Un lavage est effectué avec un tampon B (idem tampon A + 2 M NaCl) à raison de 0,3 ml/mn sur un système FPLC Pharmacia. Les différentes fractions sont récupérées en gradient de NaCl .c) Chromatography. The 70 ml of supernatant from the previous step are loaded onto a Pharmacia Heparin Sepharose column (30 ml of resin), after equilibration with buffer A (10 mM Tris-Ci pH 7.5, 0.5 mM EGTA; 5 mM MgCl2; 10 mM β-mercaptoethanol; 0.2% Triton xlOO and 10% glycerol. Washing is carried out with buffer B (same buffer A + 2 M NaCl) at a rate of 0.3 ml / min on an FPLC system Pharmacia The different fractions are recovered in NaCl gradient.
Les fractions actives ainsi récupérées sont dialysées pendant 5 h contre un tampon C: 10 mM Tris-Ci pH 7,5; 0,5 mM EGTA; 5 mM MgCl2 ; 10 mM -mercaptoéthanol; 0,2% Triton xlOO; 10% glycerol; 50 mM NaCl. Les produits issus de dialyse sont successivement chargés sur une colonne d'affinité pour les protéines se liant à l'ADN (Pharmacia, Bleue HiTrap) et élues en gradient de NaCl avec les mêmes tampons que précédemment .The active fractions thus recovered are dialyzed for 5 h against a buffer C: 10 mM Tris-Ci pH 7.5; 0.5 mM EGTA; 5 mM MgCl2; 10 mM -mercaptoethanol; 0.2% Triton x100; 10% glycerol; 50 mM NaCl. The products resulting from dialysis are successively loaded onto an affinity column for the proteins binding to DNA (Pharmacia, Blue HiTrap) and eluted in NaCl gradient with the same buffers as above.
30ml de fractions actives obtenues précédemment sont chargées sur une colonne de phosphocellulose avec de la résine Pli de Whatmann ( volume :20ml; diamètre: 2,5cm). Ces fractions ont été dialysées 5 heures contre le tampon suivant : KP04 pH7 20mM, EDTA 0,lmM, DTT ImM, Glycerol 5%, Triton X-100 0,1% et KCl 0,1M. Le débit de30ml of active fractions obtained previously are loaded onto a phosphocellulose column with Pli resin from Whatmann (volume: 20ml; diameter: 2.5cm). These fractions were dialyzed for 5 hours against the following buffer: KP04 pH7 20mM, EDTA 0, 1mM, DTT ImM, Glycerol 5%, Triton X-100 0.1% and KCl 0.1M. The flow of
FEUILLE RECTIFIEE (REGLE 91) ISAEP la colonne est réglé à 0,2ml /min, le tampon A de chargement est composé de KP04 pH7 20mM, EDTA 0,lmM, DTT ImM, Glycerol 5%, Triton X-100 0,1% et le gradient (entre 0% et 50% deB) est réalisé par le KCl présent dans tampon B à 2M.RECTIFIED SHEET (RULE 91) ISAEP the column is adjusted to 0.2 ml / min, the loading buffer A is composed of KP04 pH7 20mM, EDTA 0, lmM, DTT ImM, Glycerol 5%, Triton X-100 0.1% and the gradient (between 0% and 50% deB) is produced by KCl present in buffer B at 2M.
Ayant déjà mis en évidence que cette polymerase ne se fixe pas sur une MonoQ ou une ressource Q et ce, quel que soit le pH utilisé, nous avons essayé de la récupérer en exclusion en faisant l'hypothèse d'une fixation de l'ADN contaminant par la colonne.Having already demonstrated that this polymerase does not bind to a MonoQ or Q resource and this, whatever the pH used, we tried to recover it in exclusion by making the hypothesis of a DNA binding contaminant through the column.
Tout d'abord un essai a été réalisé en sortie de la deuxième Bleue HiTrap avec un aliquot de 5ml et dialyse selon la même méthode que pour un passage sur une Bleue HiTrap. Une deuxième tentative a été réalisée après la phosphocellulose et après deux dialyses des fractions les plus actives 45, 46 et 49. Les fractions sont tout d'abord chauffées 40 min à 85°C. Une première dialyse est alors réalisée contre le tampon KCl 0,lmM, K2HP04 1M pH7 , 5 pendant 3 heures. La deuxième dialyse est réalisée avec le tampon suivant : K2HP04 pH 7 , 5 lOmM, K2P04 lOmM, KCl 25mM, DTT ImM, Triton X-100 0,1%, Glycerol 10%, pendant 1 heure. La solution est alors chargée sur une colonne MonoQ à 0,5ml/min avec un gradient en NaCl de 0 à 20% (tampons utilisés pour l'Héparine) .First of all, a test was carried out at the end of the second HiTrap Blue with an aliquot of 5ml and dialysis according to the same method as for a passage on a HiTrap Blue. A second attempt was made after phosphocellulose and after two dialysis of the most active fractions 45, 46 and 49. The fractions are first heated 40 min at 85 ° C. A first dialysis is then carried out against the 0.1 KM KCl buffer, 1M K2HP04 pH7.5 for 3 hours. The second dialysis is carried out with the following buffer: K2HP04 pH 7.5, 10 lMM, K2PO4 lOmM, KCl 25 mM, DTT ImM, Triton X-100 0.1%, Glycerol 10%, for 1 hour. The solution is then loaded onto a MonoQ column at 0.5 ml / min with a NaCl gradient of 0 to 20% (buffers used for heparin).
6) Activités exonucléasiques .6) Exonuclease activities.
Les tests d' exonuclease 3 '-5' sont quantifiés par la libération de nucleotides marqués au P . A cette fin, une première étape permet de réaliser le marquage: l'ADN de est digéré par HindIII puis, le fragment de Klenow recopie l'ADN à partir des extrémités 3 ' -OH libres , dans un milieu contenant outre le tampon, l'ADN et l'enzyme, du 3 P dATP et du 32P dTTP, les dGTPet dCTP étant froids. Après une heure à 37°C, les quatre dNTP froids sont rajoutés en excès pour une demi- heure. La Kleno et les dNTP sont éliminés par une extraction au phénol et précipités à l'éthanol.The 3 '-5' exonuclease assays are quantified by the release of P labeled nucleotides. To this end, a first step makes it possible to carry out the labeling: the DNA of is digested with HindIII and then, the Klenow fragment copies the DNA from the free 3 '-OH ends, in a medium containing, in addition to the buffer, l DNA and the enzyme, 3 P dATP and 32 P dTTP, the dGTP and dCTP being cold. After one hour at 37 ° C, the four cold dNTPs are added in excess for half a hour. Kleno and dNTPs are removed by phenol extraction and precipitated with ethanol.
Les tests exonucléasiques sont effectués dans des solutions contenant les tampons des enzymes, 0,02mg/ml d'ADN marqué, et incubées toute la nuit à 72°C, 80°C et 95°C. Différents tampons contenant du MgCl2 ou du MnS04 sont testés. Le même test est réalisé avec la Vent en témoin positif. 101 de solution de réaction sont alors déposés sur papier DE81 (Watmann) , séchés puis comptés avant et après lavage (3 fois 5 min avec du a2HP04, 1 fois à l'eau puis à l'éthanol à 95%) en utilisant la technique de Cerenkov.The exonuclease tests are carried out in solutions containing the enzyme buffers, 0.02 mg / ml of labeled DNA, and incubated overnight at 72 ° C, 80 ° C and 95 ° C. Different buffers containing MgCl2 or MnS04 are tested. The same test is carried out with the Wind as a positive control. 101 of reaction solution are then deposited on DE81 paper (Watmann), dried and then counted before and after washing (3 times 5 min with a2HP04, 1 time with water and then with 95% ethanol) using the technique from Cerenkov.
Pour le test d'actvité exonucléasique 5'-3', la réaction de marquage utilise la polynucléotide kinase afin de marquer le substrat en 5 ' .For the 5'-3 'exonuclease activity test, the labeling reaction uses the polynucleotide kinase to label the 5' substrate.
II - Résultats .II - Results.
1) Isolement du gène de l'ADN polymerase de Thermococcus fumicolans ainsi σue des deux gènes d' intéines .1) Isolation of the DNA polymerase gene from Thermococcus fumicolans as well as from the two intein genes.
L'ADN de Thermococcus fumicolans , digéré par une série d'enzymes de restriction, a été hybride à des sondes de P. furiosus et T. li toralis, préparées par PCR et aux sondes de Pyrococcus sp. GE23 obtenues lors du clonage de cette autre ADN polymerase (Dépôt de brevet n°96 08631 auprès de l'INPI) . Comme montré dans les figures la et lb, l'hybridation de type Southern blot a révélé des fragments de deux types : un fragment HindlII- HindIII de 1,9 kb et un fragment Xhol-Xhol de 5 kb. Ces deux fragments ont été révélés uniquement avec la sondeThe DNA of Thermococcus fumicolans, digested with a series of restriction enzymes, was hybridized to probes of P. furiosus and T. li toralis, prepared by PCR and to probes of Pyrococcus sp. GE23 obtained during the cloning of this other DNA polymerase (Patent deposit n ° 96 08631 with the INPI). As shown in FIGS. 1a and 1b, the Southern blot hybridization revealed fragments of two types: a HindIII-HindIII fragment of 1.9 kb and an Xhol-Xhol fragment of 5 kb. These two fragments were revealed only with the probe
Clal-HindIII du gène de Pyrococcus sp. GE23, marquée auClal-HindIII of the Pyrococcus sp. GE23, marked with
32 P avec une exposition de deux heures. Les deux fragments repérés ont ensuite été récupérés et purifiés comme décrit précédemment dans Matériel et Méthodes puis clones dans le vecteur pUC18 déphosphorylé et digéré par32 P with a two hour exposure. The two identified fragments were then recovered and purified as described previously in Materials and Methods then cloned into the vector pUC18 dephosphorylated and digested with
HindIII ou dans le vecteur pBluescript déphosphorylé etHindIII or in the dephosphorylated pBluescript vector and
FEUILLE RECTIFIEE (REGLE 91) ISAEP digéré par Xhol . Environ 400 recombinants ( E. Coli SURE) ont été criblés avec la sonde Clal-HindIII . Sur ces 400 colonies, deux ont donné un signal positif lors de l'hybridation. (n° 9.26 et 12.79). Les deux clones ont été mis en culture en milieu LB-Amp et leurs profils de restriction étaient identiques, avec un insert de 1,8 kb. Le séquençage ultérieur de l'un de ces clones (désigné 557MACa) et la comparaison de séquence (Megalign, programme DNASTAR) ont montré qu'il correspond à la région promotrice et aux 1404 premières paires de base d'un gène d'une ADN polymerase appartenant à la famille B (2) .RECTIFIED SHEET (RULE 91) ISAEP digested with Xhol. About 400 recombinants (E. Coli SURE) were screened with the Clal-HindIII probe. Of these 400 colonies, two gave a positive signal during hybridization. (n ° 9.26 and 12.79). The two clones were cultured in LB-Amp medium and their restriction profiles were identical, with an insert of 1.8 kb. The subsequent sequencing of one of these clones (designated 557MACa) and the sequence comparison (Megalign, DNASTAR program) have shown that it corresponds to the promoter region and to the first 1404 base pairs of a DNA gene. polymerase belonging to the family B (2).
En ce qui concerne le fragment Xhol-Xhol de 5 kb, 700 recombinants ont été criblés sans aucun signal positif lors de l'hybridation. Cette absence de réussite pourrait être due à la présence d'une intéine au sein de ce fragment, le rendant alors instable dans un vecteur à haut nombre de copies de type pBluescript .With regard to the 5 kb Xhol-Xhol fragment, 700 recombinants were screened without any positive signal during hybridization. This lack of success could be due to the presence of an intein within this fragment, thus rendering it unstable in a vector with a high number of copies of pBluescript type.
Après 12 heures d'exposition, un deuxième fragment HindIII-HindIII de 2 kb a été repéré par hybridation de type Southern sur la même membrane que précédemment, en utilisant comme sonde marquée la fin du gène de Pyrococcus sp. GE23 entre les sites Xhol et SaliAfter 12 hours of exposure, a second HindIII-HindIII fragment of 2 kb was identified by Southern type hybridization on the same membrane as above, using the end of the gene for Pyrococcus sp. GE23 between the Xhol and Sali sites
(fragment obtenu par digestion de produit de PCR). Ce fragment a été clone comme précédemment. Environ 200 clones recombinants ont été criblés et quatre d'entre eux ont donné un signal positif. Les quatres clones ont un profil identique après digestion par l'enzyme de restriction HindIII. L'un d'entre eux, 557MACc, a été séquence et la comparaison de séquence a démontré qu'il s'agissait de la fin du gène de l'ADN polymerase précédemment identifiée.(fragment obtained by digestion of PCR product). This fragment was cloned as before. About 200 recombinant clones were screened and four of them gave a positive signal. The four clones have an identical profile after digestion with the restriction enzyme HindIII. One of them, 557MACc, was sequenced and the sequence comparison demonstrated that it was the end of the DNA polymerase gene previously identified.
Supposant que les fragments de gènes obtenus appartenaient à la même ADN polymerase, des oligonucléotides ont été utilisés afin d'amplifier la zone manquante. Ce fragment de PCR a ensuite été purifié sur gel comme décrit dans Matériel et Méthodes, et utilisé comme sonde marquée radioactivement pour repérer sur la membrane le fragment manquant. Après deux heures de révélation un fragment HindIII-HindIII a été révélé, de 2 kb environ. Clone comme précédemment, 600 colonies ont alors été criblées, donnant quatre clones positifs. Les quatre clones avaient le même profil et l'un d'entre eux, nommé 557MACb a été séquence et les comparaisons de séquences ont démontré qu'il s'agit de la partie intermédiaire de l'ADN polymerase et ce fragment, délimité par deux sites HindIII, s'insère parfaitement entre 557MACa et 557MACc. L'ensemble de ces trois clones donne la séquence complète de l'ADN polymerase de T. fumicolans .Assuming that the gene fragments obtained belong to the same DNA polymerase, oligonucleotides were used in order to amplify the missing area. This PCR fragment was then purified on gel as described in Materials and Methods, and used as a radioactively labeled probe to locate the missing fragment on the membrane. After two hours of development, a HindIII-HindIII fragment was revealed, of approximately 2 kb. Clone as before, 600 colonies were then screened, giving four positive clones. The four clones had the same profile and one of them, named 557MACb, was sequenced and the sequence comparisons demonstrated that it is the intermediate part of the DNA polymerase and this fragment, delimited by two HindIII sites, fits perfectly between 557MACa and 557MACc. Together, these three clones give the complete DNA polymerase sequence of T. fumicolans.
2 ) Position phylogénétiσue de Theirmococcus fumicolans2) Phylogenetic position of Theirmococcus fumicolans
Thermococcus fumicolans est une nouvelle espèce de Thermococcales décrite par Godfroy et al (8) .Thermococcus fumicolans is a new species of Thermococcales described by Godfroy et al (8).
3 ) Séquences nucléotidiαues et polypeptidiques de l'ADN polymerase de Thermococcus fumicolans .3) Nucleotide and polypeptide sequences of the DNA polymerase of Thermococcus fumicolans.
a) Séquences nucléotidicruesa) Nucleotide sequences
Les trois fragments délimités par des sites HindIII ont été assemblés (figures 2 et 3) . Ils forment à eux trois un fragment de 5039 paires de bases. Le premier fragment, issu du clone 557MACa, contient le codon de départ ATG en position 457 pb . La phase ouverte de lecture est alors ininterrompue sur 4572 paires de bases jusqu'à rencontrer un codon STOP sur le fragment issu du clone 557MACc en position 5028 pb, six paires de bases avant le dernier site HindIII. Par alignement, avec les autres gènes d'ADN polymérases disponibles en banquesThe three fragments delimited by HindIII sites were assembled (Figures 2 and 3). The three of them form a fragment of 5039 base pairs. The first fragment, coming from the clone 557MACa, contains the starting codon ATG at position 457 bp. The open reading phase is then uninterrupted on 4572 base pairs until it encounters a STOP codon on the fragment derived from the clone 557MACc in position 5028 bp, six base pairs before the last HindIII site. By alignment, with the other DNA polymerase genes available in libraries
(Pfu, Tli, GB-D, KOD, ..), avec la méthode CLUSTAL V, complétée par un alignement manuel final nécessaire pour restituer les sites d' autoépissage des intéines, deux séquences d'insertions ont été mises en évidence. - La première est insérée à la paire de base n° 1675 et se termine à 2754, étant ainsi répartie entre le fragment issu du clone 557MACa et celui du clone 557MACb.(Pfu, Tli, GB-D, KOD, ..), with the CLUSTAL V method, supplemented by a final manual alignment necessary to restore the sites of self-splicing of the inteins, two sequences of insertions were highlighted. - The first is inserted into base pair no. 1675 and ends at 2754, thus being distributed between the fragment originating from clone 557MACa and that of clone 557MACb.
- La deuxième est insérée à la base n° 3157 et se termine à 4323. Elle est, de même, répartie entre le clone 557MACb et 557MACC.- The second is inserted at the base n ° 3157 and ends at 4323. It is also distributed between the clone 557MACb and 557MACC.
Ces deux séquences d'insertions forment, avec le reste de la séquence codant pour l'ADN polymerase, un seul cadre de lecture.These two insertion sequences form, with the rest of the sequence coding for DNA polymerase, a single reading frame.
b) Séquences polypeptidiques .b) Polypeptide sequences.
La section codante du gène de l'ADN polymerase est donc constituée de trois parties disjointes. La première partie du gène, portée par 557MACa, comporte la zone codant pour 1 ' exonucléase 3'- 5', où après traduction, on reconnaît le motif FDIET. La deuxième partie, portée par 557MACb et la troisième partie portée par 557MACC comprennent les sites conservés SLYPSI, et YG.DTD. Les deux séquences d'insertion sont situées sur des zones conservées de l'ADN polymerase, la première au site DFR/SLYPSII comme I-KOD-1 de Pyrococcus sp . KOD1 et la deuxième au site D/TDG comme I-Tli-1 de T . l i toral i s . Ces deux protéines sont libérées par autoépissage proteique. Elles comportent les sites de type LAGLIDADG répétés deux fois à 100 paires de bases de distance environ. Les alignements avec les autres intéines déposées dans les banques de séquences permettent de les assimiler à des endonucléases de restriction de type archaebactérien, endonucléases qui coupent l'ADN à l'endroit précis où leur gène s'insère.The coding section of the DNA polymerase gene therefore consists of three disjoint parts. The first part of the gene, carried by 557MACa, comprises the zone coding for the 3'-5 'exonuclease, where, after translation, the motif FDIET is recognized. The second part, carried by 557MACb and the third part carried by 557MACC include the preserved sites SLYPSI, and YG.DTD. The two insertion sequences are located on conserved areas of DNA polymerase, the first at the DFR / SLYPSII site such as I-KOD-1 of Pyrococcus sp. KOD1 and the second at the D / TDG site as T-I-Tli-1. l i toral i s. These two proteins are released by protein self-splicing. They include LAGLIDADG type sites repeated twice at approximately 100 base pairs apart. The alignments with the other inteins deposited in the sequence banks make it possible to assimilate them to restriction endonucleases of the archaebacterial type, endonucleases which cut DNA at the precise place where their gene is inserted.
c) Comparaison avec les autres séquences d'ADN polymérases . L'alignement des différentes séquences polypeptidiques des ADN polymérases de Thermococcales disponibles en banque, P. abyssi strain GE5 , Pyrococcus sp. GE23, Pyrococcus sp . KOD1 , Pyrococcus sp. GB-D, P. furi os us , Pyrococcus sp 9°N, T. l i toralis avec la séquence de T. fumicolans (sans intéines), réalisé avec le programme CLUSTAL utilisant la matrice PAM250, donne les niveaux de similarité entre ces diverses polymérases figurant au tableau 1 ci-dessous.c) Comparison with other DNA polymerase sequences. The alignment of the different polypeptide sequences of the DNA polymerases of Thermococcales available in the bank, P. abyssi strain GE5, Pyrococcus sp. GE23, Pyrococcus sp. KOD1, Pyrococcus sp. GB-D, P. furi os us, Pyrococcus sp 9 ° N, T. li toralis with the sequence of T. fumicolans (without inteins), carried out with the CLUSTAL program using the PAM250 matrix, gives the levels of similarity between these various polymerases listed in Table 1 below.
Tableau 1Table 1
Figure imgf000026_0001
Figure imgf000026_0001
Le niveau de similarité le plus proche est celui observé avec Pyrococcus sp . 9°N (90,6%), ce qui indique clairement que l'ADN polymerase de T. fumicolans est originale au niveau de sa séquence et constitue de ce fait une nouvelle ADN polymerase.The closest similarity level is that observed with Pyrococcus sp. 9 ° N (90.6%), which clearly indicates that the DNA polymerase of T. fumicolans is original in terms of its sequence and therefore constitutes a new DNA polymerase.
d) Comparaison des intéines avec les autres intéines disponibles dans les banques .d) Comparison of the inteins with the other inteins available in banks.
Les séquences disponibles en banque ont été alignées selon les mêmes méthodes que précédemment. Les comparaisons de l'ensemble des intéines démontrent que celles-ci sont réparties en trois groupes correspondant aux sites d'insertion des motifs A (R/SLYPSI), B (KILAN/S) et C (D/TDG) . L'analyse des niveaux de similarité et la recherche de relations phylogénétiques n'ont de sens que pour des intéines appartenant à la même classe, c'est à dire s ' insérant dans un motif donné. Les niveaux de similarités pour I-Tfu-1 (classe A) et I-Tfu-2 (classe C) avec leurs intéines homologues déjà décrites sont donnés respectivement aux tableaux 2 et 3 ci- dessous . Tableau 2The sequences available in the bank were aligned using the same methods as above. The comparisons of all the inteins show that these are divided into three groups corresponding to the sites of insertion of the motifs A (R / SLYPSI), B (KILAN / S) and C (D / TDG). The analysis of the levels of similarity and the search for phylogenetic relationships only make sense for inteins belonging to the same class, that is to say, fitting into a given motif. The similarity levels for I-Tfu-1 (class A) and I-Tfu-2 (class C) with their homologous inteins already described are given in Tables 2 and 3 respectively below. Table 2
FEUILLE RECTIFIEE (REGLE 91) ISA EP
Figure imgf000027_0001
RECTIFIED SHEET (RULE 91) ISA EP
Figure imgf000027_0001
Tableau 3Table 3
Figure imgf000027_0002
Figure imgf000027_0002
I-Tfu-1 et I-Tfu-2 semblent représenter deux nouveaux "allèles" des "homing" endonucléases archaebactériennes des classes A et C.I-Tfu-1 and I-Tfu-2 seem to represent two new "alleles" of "homing" archaebacterial endonucleases of classes A and C.
I-Tfu-1 est le troisième allèle connu d' intéine s ' insérant au site A des ADN polymerase d'Archaea, tandis que I-Tfu-2 est le second de sa classe.I-Tfu-1 is the third known allele of intein inserting into the A site of Archaea DNA polymerase, while I-Tfu-2 is the second of its class.
5 ) Expression, caractérisation et activité de l'ADN polymerase de T. fumicolans .5) Expression, characterization and activity of T. fumicolans DNA polymerase.
a) Clonaσe et expression. Un insert de 4595 pb obtenu par PCR longue- distance avec les amorces TfuDir et TfuRev et couvrant la totalité du gène de l'ADN polymerase de Thermococcus fumicolans, avec les deux intéines, a été clone aux sites Ndel et BamHI d'un vecteur pour transformer la souche E. coli Novablue. Des mini-préparations d'ADN plasmidique ont été réalisées sur une dizaine de clones transformants et ont toutes donné un signal positif à l'hybridation. Deux clones ont été retenus sur la base de leur profil après une digestion par Ndel et Sali ou par HindIII. Ces deux plasmides ont ensuite été transformés dans la souche E. coli BL21(DE3)pLysS.a) Clonaσe and expression. A 4595 bp insert obtained by long-distance PCR with the primers TfuDir and TfuRev and covering the entire DNA polymerase gene of Thermococcus fumicolans, with the two inteins, was cloned at the NdeI and BamHI sites of a vector for transform the E. coli Novablue strain. Mini-plasmid DNA preparations were carried out on ten transforming clones and all gave a positive signal to hybridization. Two clones were selected on the basis of their profile after digestion with Ndel and Sali or with HindIII. These two plasmids were then transformed into the E. coli BL21 (DE3) pLysS strain.
Des essais d'expression ont été réalisés en culture de 5 ml afin de déterminer les conditions optimales de culture et d'induction. Tout d'abord les essais de cultures ont été réalisés à 37°C, où l'on observe une lyse cellulaire trop importante, puis à 30°C où la culture ne lyse pratiquement pas. La culture est réalisée en milieu 2xYT, où le rendement de production est meilleur et la lyse réduite, supplémenté avec de l'ampicilline (lOOμg/ml) et du chloramphenicol (15μg/ml) . L'expression est alors induite en phase exponentielle de croissance (DO600nm = 0,6 à 0,7) avec une concentration en IPTG de ImM, concentration qui s'est avérée optimale. Des échantillons sont prélevés avant induction puis 2 heures, 4 heures et 6 heures après induction, ainsi qu'après une nuit. Des prélèvemments sont aussi réalisés sur les cultures non induites après 6, 8, 12 et 24 heures de culture .Expression tests were carried out in 5 ml culture in order to determine the optimal culture and induction conditions. First of all, the culture tests were carried out at 37 ° C., where there is too much cell lysis, then at 30 ° C. where the culture practically does not lyse. Culture is carried out in a 2xYT medium, where the production yield is better and the lysis reduced, supplemented with ampicillin (100 μg / ml) and chloramphenicol (15 μg / ml). Expression is then induced in the exponential growth phase (OD600nm = 0.6 to 0.7) with an IPTG concentration of ImM, a concentration which has been found to be optimal. Samples are taken before induction, then 2 hours, 4 hours and 6 hours after induction, as well as overnight. Samples are also taken from non-induced cultures after 6, 8, 12 and 24 hours of culture.
Les échantillons sont alors traités comme indiqué au chapitre Matériel et Méthodes, afin de tester le niveau d'activité de l'ADN polymerase recombinante parThe samples are then treated as indicated in the Materials and Methods chapter, in order to test the level of activity of the recombinant DNA polymerase by
39 incorporation de thymidine tritiée ou de P dCTP. Cette première technique, qui permet une approche qualitative, nous donne deux types de comportement. Un des clones est non inductible et la meilleure activité est obtenue après une nuit de culture (clone rTful-1) . Un second clone est inductible et présente une activité maximale après quatre heures d'induction, activité qui diminue par la suite39 incorporation of tritiated thymidine or P dCTP. This first technique, which allows a qualitative approach, gives us two types of behavior. One of the clones is non-inducible and the best activity is obtained after a night of culture (rTful-1 clone). A second clone is inducible and exhibits maximum activity after four hours of induction, an activity which subsequently decreases
(clone rTful00-2). Deux autres clones, aussi testés sont faiblement inductibles et expriment très peu l'ADN polymerase . Les clones rTful-1 et rTful00-2 sont ensuite testés en erlenmeyer de 50 ml dans les conditions décrites précédemment. Seul le clone inductible rTfulOO-2 a une expression constante en volume de 50 ml. La suite des travaux a donc été réalisée sur ce clone .(rTful00-2 clone). Two other clones, also tested, are weakly inducible and express very little DNA polymerase. The rTful-1 and rTful00-2 clones are then tested in a 50 ml Erlenmeyer flask under the conditions described above. Only the inducible rTfulOO-2 clone has a constant volume expression of 50 ml. The rest of the work was therefore carried out on this clone.
b) Fermentation et extraction des cellules .b) Fermentation and extraction of cells.
La culture du clone Tful00-2 à été réalisée dans un fermenteur de 16 litres, dans le milieu 2xYT supplémenté en ampicilline et chloramphenicol. La préculture, de 750ml a été réalisée la veille et arrêtée en phase exponentielle (DO 600nm= 0,7-0,8) et laissée àThe culture of the clone Tful00-2 was carried out in a 16 liter fermenter, in the 2xYT medium supplemented with ampicillin and chloramphenicol. The 750 ml preculture was carried out the day before and stopped in the exponential phase (OD 600nm = 0.7-0.8) and left at
4°C pour la nuit. Le fermenteur a été préparé et mis en température à 30°C avec le milieu de culture. La préculture a été remise à 30°C une heure avant son transfert dans le fermenteur. Le volume final de culture est de 15 litres. Les conditions sont les suivantes: température = 30°C ; agitation = 300 rpm. L'induction avec 1 mM d'IPTG a été réalisée à DO 600 = 0,58. Le pH de la culture a été régulé à 7 pendant la phase d'acidification puis laissé libre lors de la phase alcaline. Les bactéries ont été prélevées après quatre heures d'induction, alors que le pH était de 8,3. La culture a ensuite été centrifugée puis les cellules ont été divisées en trois lots. L'un d'eux, 20 g de pâte, a été repris dans 80 ml de tampon de lyse pour un traitement ultérieur indiqué au chapitre Matériel et méthodes .4 ° C for the night. The fermenter has been prepared and put in temperature at 30 ° C with the culture medium. The preculture was returned to 30 ° C one hour before its transfer to the fermenter. The final culture volume is 15 liters. The conditions are as follows: temperature = 30 ° C; agitation = 300 rpm. Induction with 1 mM IPTG was carried out at OD 600 = 0.58. The pH of the culture was adjusted to 7 during the acidification phase and then left free during the alkaline phase. The bacteria were removed after four hours of induction, when the pH was 8.3. The culture was then centrifuged and the cells were divided into three batches. One of them, 20 g of paste, was taken up in 80 ml of lysis buffer for further processing indicated in the Materials and methods chapter.
c) Purification de l'ADN polymerase de T. fumicolans .c) Purification of T. fumicolans DNA polymerase.
La purification a été .effectuée comme indiqué précédemment (Matériel et Méthodes) . Pour la colonne Héparine -Sépharose, un gradient de 3 à 50% de tampon B, correspondant aux volume 365 ml à 1363 ml, permet de récupérer 73 fractions de 6 ml . Le pic d'activité de la polymerase est obtenu pour une valeur de gradient de 0,5 M environ, et correspond aux fractions 55/56 (dosées à 10 et 12 unités respectivement) comme indiqué à la figure 7. Ces fractions, incubées à 37°C pendant la nuit en présence d'ADN de pBR322, dégradent l'ADN et présentent en conséquence des traces de nucléase de l'hôte, non visibles sur gel. Leur élimination, ou tout au moins une réduction substancielle de leur concentration a été réalisée par un passage sur colonne d'affinité (Bleue Hitrap) .The purification was carried out as indicated previously (Materials and Methods). For the Heparin-Sepharose column, a gradient of 3 to 50% of buffer B, corresponding to the volume 365 ml to 1363 ml, makes it possible to recover 73 fractions of 6 ml. The peak of polymerase activity is obtained for a gradient value of approximately 0.5 M, and corresponds to fractions 55/56 (assayed at 10 and 12 units respectively) as indicated in FIG. 7. These fractions, incubated at 37 ° C overnight in the presence of pBR322 DNA, degrade the DNA and consequently show traces of host nuclease, not visible on gel. Their elimination, or at least a substantial reduction in their concentration, was achieved by passing through an affinity column (Blue Hitrap).
Les fractions 54 à 60, regroupées et dialysées, sont chargées à raison de 0,25 ml/mn. L'élution permet de récupérer 65 fractions de 5 ml avec des pics d'activité pour les fractions 36 à 56. Le dosage de l'activité fait apparaître une concentration de 3 à 5 unités pour les fractions 36 à 40. Ces fractions, mises en présence d'ADN à 37°C, pressentent une faible activité nucléasique. Néanmoins, l'activité sur le plasmide pBR322 àFractions 54 to 60, grouped and dialyzed, are loaded at a rate of 0.25 ml / min. The elution makes it possible to recover 65 fractions of 5 ml with peaks of activity for fractions 36 to 56. The dosage of the activity shows a concentration of 3 to 5 units for fractions 36 to 40. These fractions, when put in the presence of DNA at 37 ° C, sense a weak nucleic activity. Nevertheless, the activity on the plasmid pBR322 to
37°C toute la nuit montre une nette amélioration de la pureté de l'enzyme. Une deuxième colonne Bleue HiTrap a été réutilisée en prenant les fractions 36 à 44 et dialysées comme précédemment. 25ml sur les 30ml de fraction ont été injectés sur la colonne avec un débit de 0,25ml/min (5 ml étant gardés pour essayer une MonoQ) . Après cette deuxième colonne Bleue HiTrap, l'activité sur le plasmide pBR322 fermé et incubé toute la nuit avec des fractions de la colonne, est nulle. L'incubation d'une heure à 72°C de fractions avec l'ADN de Lambda digéré par HindIII montre une très nette dégradation, mettant en évidence l'activité exonucléase 3 '-5' associée à notre ADN polymerase. Le pic d'activité se situe entre les fractions 27 et 32. L'ADN polymerase plus pure est donc sortie plus tôt sur le gradient de NaCl. Sur les fractions les plus actives, un comptage a été réalisé donnant une activité supérieure à 5U/μl pour les fractions 29, 30 et 31. La figure 4 montre le résultat sur gel SDS-PAGE. Suite à ces trois colonnes, la pureté de la polymerase est nettement améliorée. Néanmoins, il reste des traces d'ADN de E. coli fixé à la polymerase et mises en évidence par PCR. Deux colonnes supplémentaires vont donc être mis en oeuvre. 30ml de fractions actives obtenues précédemment sont chargées sur une colonne de phosphocellulose (celle-ci devrait fixer différemment l'ADN et la polymerase). L'activité de la polymerase est repérée par son activité exonucléasique 3 '-5' élevée à 72°C sur l'ADN de Lambda digéré par HindIII. L'activité la plus forte se situe entre les fractions 40 et 49 avec un pic net en 46 et 47. Sur ces fractions 40 à 49 l'ADN de pBR322 est intact après une nuit à 37°C. La figure 5 nous montre les résultats sur gel avec des activités37 ° C overnight shows a marked improvement in the purity of the enzyme. A second HiTrap Blue column was reused, taking fractions 36 to 44 and dialyzed as before. 25ml of the 30ml fraction were injected onto the column with a flow rate of 0.25ml / min (5ml being kept to try a MonoQ). After this second Blue HiTrap column, the activity on the plasmid pBR322 closed and incubated overnight with fractions of the column is zero. The one hour incubation at 72 ° C of fractions with the DNA of Lambda digested with HindIII shows a very clear degradation, highlighting the exonuclease activity 3 '-5' associated with our DNA polymerase. The peak of activity is between fractions 27 and 32. The purer DNA polymerase therefore left earlier on the NaCl gradient. On the most active fractions, a count was made giving an activity greater than 5U / μl for fractions 29, 30 and 31. FIG. 4 shows the result on SDS-PAGE gel. Following these three columns, the purity of the polymerase is significantly improved. Nevertheless, traces of DNA from E. coli attached to the polymerase and demonstrated by PCR remain. Two additional columns will therefore be implemented. 30ml of active fractions obtained previously are loaded onto a phosphocellulose column (this should fix the DNA and the polymerase differently). The activity of the polymerase is identified by its high 3 '-5' exonuclease activity at 72 ° C. on the DNA of Lambda digested with HindIII. The strongest activity is located between fractions 40 and 49 with a sharp peak at 46 and 47. On these fractions 40 to 49 the pBR322 DNA is intact after overnight at 37 ° C. Figure 5 shows us the results on gel with activities
FEUILLE RECTIFIEE (REGLE 91) ISAEP mesurées de 6U/μl pour la fraction 46, 4,5U/μl pour la 47 et 3U/μl pour la 48. Néanmoins il reste encore des traces d'ADN de E. coli .RECTIFIED SHEET (RULE 91) ISAEP measured at 6U / μl for fraction 46, 4.5U / μl for 47 and 3U / μl for 48. However, traces of DNA from E. coli still remain.
Ayant déjà mis en évidence que cette polymerase ne se fixe pas sur une MonoQ ou une ressource Q et ce quel que soit le pH utilisé, nous avons essayé de la récupérer en exclusion en espérant une fixation de l'ADN contaminant par la colonne.Having already demonstrated that this polymerase does not bind to a MonoQ or Q resource, regardless of the pH used, we tried to recover it by exclusion, hoping for fixation of the contaminating DNA by the column.
Tout d'abord un essai a été réalisé en sortie de la deuxième Bleue HiTrap avec 5ml dialyses Les résultats ont été décevants, les fractions d'exclusion étant peu actives en incorporation.First of all, a test was carried out at the end of the second HiTrap Blue with 5 ml dialysis. The results were disappointing, the exclusion fractions being not very active in incorporation.
Une deuxième tentative a été réalisée après la phosphocellulose et après deux dialyses des fractions les plus actives 45, 46 et 49. Les fractions sont tout d'abord chauffées 40 min à 85°C en raison d'une détection de contaminant dégradant le pBR322 à 72°C en une nuit. Aucune floculation n'est alors visible et l'extrait est mis sur glace. Un nouveau test démontre que la contamination semble réduite. Après les dialyses et le passage sur colonne, l'activité exonucléasique est mise en évidence sur les fractions d'exclusion 3 à 7. L'activité est dosée à 2U/μl et l'enzyme est visualisée sur la figure 10. Suite a cette dernière étape de purification, nous avons obtenu des résultats positifs en PCR et comparables à ceux obtenus pour la Vent.A second attempt was made after phosphocellulose and after two dialyses of the most active fractions 45, 46 and 49. The fractions are first heated 40 min at 85 ° C. due to the detection of a contaminant degrading pBR322 at 72 ° C overnight. No flocculation is then visible and the extract is put on ice. A new test shows that the contamination seems reduced. After the dialysis and the passage through a column, the exonuclease activity is demonstrated on the exclusion fractions 3 to 7. The activity is assayed at 2U / μl and the enzyme is displayed in FIG. 10. Following this last purification step, we obtained positive PCR results and comparable to those obtained for Wind.
d) Caractérisation des activité des fractions purifiées . L'activité de l'ADN polymerase des différentesd) Characterization of the activities of the purified fractions. DNA polymerase activity of different
32 fractions a été dosée par incorporation au P dATP selon le protocole décrit en Matériel et Méthodes.32 fractions were assayed by incorporation into P dATP according to the protocol described in Materials and Methods.
- Amplification de gènes in vi tro . Un fragment de 459 pb a été amplifié à partir d'un ADN genomique d'Archaebactérie ( Thermococcus sp. GE- Amplification of genes in vi tro. A 459 bp fragment was amplified from genomic DNA from Archaebacteria (Thermococcus sp. GE
FEUILLE RECTIFIEE (REGLE 91) ISA/EP 8) avec des amorces spécifiques. Différents tampons ont été utilisés:RECTIFIED SHEET (RULE 91) ISA / EP 8) with specific primers. Different buffers were used:
- tampon lOx R: Tris HC1 pH 8,8: 300mM; KCl: 500mM; MgC12: 30mM; T een 20: 0,1% - tampon lOx H: Tris HC1 : pH 8,8: 300 mM; KCl:- Ox R buffer: Tris HC1 pH 8.8: 300mM; KCl: 500mM; MgC12: 30mM; T een 20: 0.1% - 10 × H buffer: Tris HC1: pH 8.8: 300 mM; KCl:
500mM; MgC12 : 15mM; Tween 20: 0,1%500mM; MgC12: 15mM; Tween 20: 0.1%
- tampon lOx T: Tris HC1 pH 8,8: 600mM; KCl: 500mM; MgC12 : 15mM; Tween 20: 0,1%- lOx T buffer: Tris HC1 pH 8.8: 600mM; KCl: 500mM; MgC12: 15mM; Tween 20: 0.1%
- tampon lOx S: Tris Hcl pH 8,8: 200mM; KCl: 250mM; MgC12 : 20mM; Tween 20: 0,1%- lOx S buffer: Tris Hcl pH 8.8: 200mM; KCl: 250mM; MgC12: 20mM; Tween 20: 0.1%
Trente cycles ont été réalisés, chacun comprenant une étape de dénaturation à 94°C pendant 30 sec., une étape d'hybridation des amorces à 51°C pendant 1 min. puis une étape d' élongation à 72°C pendant 2 min. La figure 8 présente les résultats obtenus avec un volume reactionnel de 50 μl pour des quantités d'ADN polymerase de Thermococcus fumicolans de 2,7 unités. En l'état actuel, les meilleurs résultats avec la Tfu sont obtenus avec le tampon R . La figure 9 représente les résultats de l'amplification d'un fragment de 1,6 kb avec la Tfu purifiée sur colonnes d'héparine puis de séphacryl-bleue, et un tampon reactionnel lOx ayant la composition suivante: Tris HC1 pH 8,8: 200mM; KCl: lOOmM; (NH4)2Sθ4: lOOmM; MgS04 : 20mM; Triton X-100 : 1%.Thirty cycles were carried out, each comprising a denaturation step at 94 ° C for 30 sec., A step of hybridization of the primers at 51 ° C for 1 min. then an elongation step at 72 ° C for 2 min. FIG. 8 presents the results obtained with a reaction volume of 50 μl for quantities of DNA polymerase from Thermococcus fumicolans of 2.7 units. As it stands, the best results with Tfu are obtained with buffer R. FIG. 9 represents the results of the amplification of a 1.6 kb fragment with the purified Tfu on heparin and then sephacryl-blue columns, and a 10 × reaction buffer having the following composition: Tris HC1 pH 8.8 : 200mM; KCl: 100 mM; (NH4) 2Sθ4: 100mM; MgSO4: 20mM; Triton X-100: 1%.
- Activité exonucléasique.- Exonuclease activity.
Les tests d'activité, selon le protocole détaillé en Matériels et Méthodes, ne révèlent pas d'activité exonucléasique 5 '-3' chez la Tfu. Ceci est en conformité avec la structure de l'enzyme déduite de l'analyse de la séquence polypeptidique qui ne fait pas apparaître de domaine fonctionnel exonucléase 5 '-3', contrairement à ce qui est observé pour DNA poli de E. coli et la Taq.The activity tests, according to the protocol detailed in Materials and Methods, do not reveal any 5 '-3' exonuclease activity in Tfu. This is in accordance with the structure of the enzyme deduced from the analysis of the polypeptide sequence which does not show a functional domain of exonuclease 5 '-3', contrary to what is observed for polished DNA of E. coli and the Taq.
Les tests d'activité, selon le protocole détaillé au chapitre Matériels et Méthodes, font apparaître une activité exonucléasique 3 '-5' (activité proof-reading ou de correction d'erreurs) chez la Tfu, à un niveau sensiblement égal à celui de la Vent comme montré dans le tableau 4 ci-dessous. Le tableau 4 rapporte la mesure de l'actvité exonucléasique 3 '-5' de la Tfu en fonction de la concentration en dNTP, et en comparaison avec la Vent.The activity tests, according to the protocol detailed in the Materials and Methods chapter, a 3 '-5' exonuclease activity (proof-reading or error correction activity) appears in the Tfu, at a level substantially equal to that of the Wind as shown in table 4 below. Table 4 reports the measurement of the 3 '-5' exonuclease activity of Tfu as a function of the dNTP concentration, and in comparison with the Wind.
Tableau 4Table 4
Figure imgf000033_0001
Ces résultats sont en conformité avec la structure de l'enzyme déduite de l'analyse de la séquence polypeptidique qui révèle la présence d'un domaine exonucléasique 3 '-5' en position N-terminale, ainsi que la présence des motifs catalytiques caractéristiques de ce domaine. La Tfu est sensible à une concentration de l'ordre de 0 , 8 mM de dNTP, tandis que la Vent manifeste une sensibilité dès 0,5 mM. Cette activité est connue pour améliorer in vi tro la fidélité des polymérases utilisées en PCR. En outre, cette activité exonucléasique est confirmée par un test plus simple. Les fractions purifiées, dépourvues d'activité nucléasique à 37°C, mises en présence d'ADN de digéré par HindIII puis exposé à 72°C pendant la nuit, dégradent complètement cet ADN, mettant ainsi en évidence la présence de l'activité 3 '-5' exonucléase de la T f u ainsi que sa thermostabilité .
Figure imgf000033_0001
These results are in accordance with the structure of the enzyme deduced from the analysis of the polypeptide sequence which reveals the presence of a 3 '-5' exonuclease domain in the N-terminal position, as well as the presence of the catalytic motifs characteristic of this domain. The Tfu is sensitive to a concentration of the order of 0.8 mM of dNTP, while the Wind manifests a sensitivity from 0.5 mM. This activity is known to improve in vi tro the fidelity of the polymerases used in PCR. In addition, this exonuclease activity is confirmed by a simpler test. The purified fractions, devoid of nuclease activity at 37 ° C., placed in the presence of DNA digested with HindIII and then exposed to 72 ° C. overnight, completely degrade this DNA, thus demonstrating the presence of activity 3 '-5' exonuclease of T fu and its thermostability.
- Thermostabilité.- Thermostability.
FEUILLE RECTIFIEE (REGLE 91) ISA/EP 32RECTIFIED SHEET (RULE 91) ISA / EP 32
La thermostabilité mesurée selon le protocole décrit précédemment (Matériel et Méthodes), ou mieux, l'activité résiduelle en incorporation à 72°C après exposition de l'enzyme à des températures élevées pendant des temps variables, est donnée dans le tableau 5 ci- dessous .The thermostability measured according to the protocol described above (Materials and Methods), or better, the residual activity in incorporation at 72 ° C. after exposure of the enzyme to high temperatures for variable times, is given in table 5 below. below.
Tableau 5Table 5
Figure imgf000034_0001
Figure imgf000034_0001
Cette thermostabilité, inférieure à celle des polymérases issues d'organismes plus hyperthermophiles tels que les Pyrococcus , n'en demeure pas moins très largement supérieure à celles des polymérases issues des Thermus et en particulier toutes les Taq. La thermostabilité de l'enzyme recombinante purifiée, tant pour le domaine polymerase que pour l' exonucléase, est de toute manière très largement supérieure à tous les besoins connus en PCR. This thermostability, which is lower than that of polymerases from more hyperthermophilic organisms such as Pyrococcus, is nonetheless very much greater than that of polymerases from Thermus and in particular all Taq. The thermostability of the purified recombinant enzyme, both for the polymerase domain and for the exonuclease, is in any case very much higher than all known requirements in PCR.
REFERENCES BIBLIOGRAPHIQUES .BIBLIOGRAPHICAL REFERENCES.
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F. B. Perler, and M. Q. Xu. 1996. Protein splicing involving the Saccharomyces cerevisiae VMA intein - The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing System. Journal of Biological Chemistry.F. B. Perler, and M. Q. Xu. 1996. Protein splicing involving the Saccharomyces cerevisiae VMA intein - The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing System. Journal of Biological Chemistry.
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Thermococcus sp . 9°N-7 and mutations affecting 3 ' -5 ' exonucléase activity. Proc. Ntl . Acad. Sci. USA. 93:5281- 5285.Thermococcus sp. 9 ° N-7 and mutations affecting 3 '-5' exonuclease activity. Proc. Ntl. Acad. Sci. USA. 93: 5281-5285.
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Cloning and sequencing of the gène. European Journal of Biochemistry. 194:839-844. 18. Uemori, T., Y. Ishino, H. Toh, K. Asada, and I. Kato. 1993. Organization and nucleotide séquence of the DNA polymerase gène from the archaeon Pyrococcus furiosus . Nucleic Acids Research. 21(2) :259- 265. Cloning and sequencing of the gene. European Journal of Biochemistry. 194: 839-844. 18. Uemori, T., Y. Ishino, H. Toh, K. Asada, and I. Kato. 1993. Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus. Nucleic Acids Research. 21 (2): 259-265.
LISTE DE SEQUENCESLIST OF SEQUENCES
;i) INFORMATION GÉNÉRALES :; i) GENERAL INFORMATION:
(i) DEPOSANT : APPLIGENE - ONCOR(i) DEPOSITOR: APPLIGENE - ONCOR
( ii) TITRE DE L' INVENTION: ADN POLYMERASE THERMOSTABLE D'ARCHAEBACTÉRIES DE L 'ESPECE THERMOCOCCUS fumicolans (iii) NOMBRE DE SEQUENCES: 4(ii) TITLE OF THE INVENTION: THERMOSTABLE DNA POLYMERASE OF ARCHAEBACTERIA OF THE THERMOCOCCUS fumicolans SPECIES (iii) NUMBER OF SEQUENCES: 4
(2) INFORMATION POUR LA SEQ ID NO : 1 :(2) INFORMATION FOR SEQ ID NO: 1:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 5039 paires de bases(A) LENGTH: 5039 base pairs
(B) TYPE: nucleotide(B) TYPE: nucleotide
(C) NOMBRE DE BRIN: double(C) NUMBER OF STRANDS: double
(D) CONFIGURATION: linéaire (ii) TYPE DE MOLECULE: ADN(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA
(ix) CARACTERISTIQUES(ix) CHARACTERISTICS
(A) NOM/CLE: séquence codante de l 'ADN polymerase de THERMOCOCCUS fumicolans Tfu(A) NAME / KEY: DNA polymerase sequence of THERMOCOCCUS fumicolans Tfu
(B) EMPLACEMENT: de 457 à 5028 ( ix ) CARACTERI STIQUES(B) LOCATION: from 457 to 5028 (ix) CHARACTERISTICS
(A) NOM/CLE: séquence codante de 1 ' intéine I-Tfu-1(A) NAME / KEY: coding sequence of the I-Tfu-1 intein
(B) EMPLACEMENT: de 1675 à 2754 ( ix ) CARACTERI STIQUES(B) LOCATION: from 1675 to 2754 (ix) CHARACTERISTICS
(A) NOM/CLE: séquence codante de 1 ' intéine I-Tfu-2(A) NAME / CLE: coding sequence of the I-Tfu-2 intein
(B) EMPLACEMENT: de 3157 à 4323 ( ix ) CARACTERI STIQUES(B) LOCATION: from 3157 to 4323 (ix) CHARACTERISTICS
(A) NOM/CLE: codon stop(A) NAME / KEY: stop codon
(B) EMPLACEMENT: de 5026 à 5028(B) LOCATION: 5026 to 5028
(xi) DESCRIPTION DE LA SEQUENCES: SEQ ID NO:l :(xi) DESCRIPTION OF THE SEQUENCES: SEQ ID NO: l:
AGCTTAAAGC GTCCGCCACT ACTTCCTGAA AGCTCACGCG GTAAAACAGC TCCATGCTCG 60AGCTTAAAGC GTCCGCCACT ACTTCCTGAA AGCTCACGCG GTAAAACAGC TCCATGCTCG 60
GCTCTTCGAT GGGAGGTTTA AAAAGGTGGT GGTGAGGTTT ATTAGGAAGA AGGCTCAACT 120GCTCTTCGAT GGGAGGTTTA AAAAGGTGGT GGTGAGGTTT ATTAGGAAGA AGGCTCAACT 120
AGAGACGGTG GGAGTATGGA AGAGGTCGAC AGGCTCGTGT TCAACTTTCC CCTCTTCAAA 180AGAGACGGTG GGAGTATGGA AGAGGTCGAC AGGCTCGTGT TCAACTTTCC CCTCTTCAAA 180
GATTACTGGG AAAAGGAGCG GTTCCTCAAG GTCGTTGGGC TTCTGGTGAG CCACCAGATA 240GATTACTGGG AAAAGGAGCG GTTCCTCAAG GTCGTTGGGC TTCTGGTGAG CCACCAGATA 240
ACGTTTGAGA AAGCTGCCGA GCTTCTGGAC ATGAGGCTCG AAGAGCTGGC GTTCCTCCTT 300ACGTTTGAGA AAGCTGCCGA GCTTCTGGAC ATGAGGCTCG AAGAGCTGGC GTTCCTCCTT 300
GACAAGCTCG GCGTTGAGTA CTCGCTTCTT GATGATGAAG AGGCCAGACT TGAGAGAGAA 360GACAAGCTCG GCGTTGAGTA CTCGCTTCTT GATGATGAAG AGGCCAGACT TGAGAGAGAA 360
GAGGCCAATA AGCTCATGGG GGAAATGAAG GGTGGAGCGT TTGTCTGATT CTTCTGAGCT 420GAGGCCAATA AGCTCATGGG GGAAATGAAG GGTGGAGCGT TTGTCTGATT CTTCTGAGCT 420
GTTATTGGTG TTTCACAGGC TGGGAGGTGG TGGATT ATG ATC CTC GAT ACA GAC 474GTTATTGGTG TTTCACAGGC TGGGAGGTGG TGGATT ATG ATC CTC GAT ACA GAC 474
Met Ile Leu Asp Thr Asp 1 5Met Ile Leu Asp Thr Asp 1 5
TAC ATC ACC GAA GAC GGA AGG CCC GTC ATC AGG GTG TTC AAG AAG GAG 522TAC ATC ACC GAA GAC GGA AGG CCC GTC ATC AGG GTG TTC AAG AAG GAG 522
Tyr Ile Thr Glu Asp Gly Arg Pro Val Ile Arg Val Phe Lys Lys Glu 10 15 20 AAC GGC GAG TTC AAA ATC GAG TAC GAC AGG GAC TTC GAG CCT TAC ATC 570 Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg Asp Phe Glu Pro Tyr Ile 25 30 35Tyr Ile Thr Glu Asp Gly Arg Pro Val Ile Arg Val Phe Lys Lys Glu 10 15 20 AAC GGC GAG TTC AAA ATC GAG TAC GAC AGG GAC TTC GAG CCT TAC ATC 570 Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg Asp Phe Glu Pro Tyr Ile 25 30 35
TAC GCT CTC CTG AAG GAC GAT TCC GCG ATC GAG GAC GTC AAG AAG ATA 618 Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile Glu Asp Val Lys Lys Ile 40 45 50TAC GCT CTC CTG AAG GAC GAT TCC GCG ATC GAG GAC GTC AAG AAG ATA 618 Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile Glu Asp Val Lys Lys Ile 40 45 50
ACT GCA AGC CGG CAC GGT ACC ACC GTC AGG GTC GTC AGG GCC GGG AAG 666 Thr Ala Ser Arg His Gly Thr Thr Val Arg Val Val Arg Ala Gly Lys 55 60 65 70ACT GCA AGC CGG CAC GGT ACC ACC GTC AGG GTC GTC AGG GCC GGG AAG 666 Thr Ala Ser Arg His Gly Thr Thr Val Arg Val Val Arg Ala Gly Lys 55 60 65 70
GTG AAG AAG AAG TTC CTC GGC AGG CCG ATA GAG GTC TGG AAG CTC TAC 714 Val Lys Lys Lys Phe Leu Gly Arg Pro Ile Glu Val Trp Lys Leu Tyr 75 80 85GTG AAG AAG AAG TTC CTC GGC AGG CCG ATA GAG GTC TGG AAG CTC TAC 714 Val Lys Lys Lys Phe Leu Gly Arg Pro Ile Glu Val Trp Lys Leu Tyr 75 80 85
TTC ACC CAT CCC CAG GAC GTT CCG GCA ATC AGG GAC AAA ATC AGG GAG 762 Phe Thr His Pro Gin Asp Val Pro Ala Ile Arg Asp Lys Ile Arg Glu 90 95 100TTC ACC CAT CCC CAG GAC GTT CCG GCA ATC AGG GAC AAA ATC AGG GAG 762 Phe Thr His Pro Gin Asp Val Pro Ala Ile Arg Asp Lys Ile Arg Glu 90 95 100
CAC CCT GCC GTG GTC GAC ATA TAT GAG TAC GAC ATA CCC TTT GCC AAG 810 His Pro Ala Val Val Asp Ile Tyr Glu Tyr Asp Ile Pro Phe Ala Lys 105 110 115CAC CCT GCC GTG GTC GAC ATA TAT GAG TAC GAC ATA CCC TTT GCC AAG 810 His Pro Ala Val Val Asp Ile Tyr Glu Tyr Asp Ile Pro Phe Ala Lys 105 110 115
CGC TAC CTC ATC GAT AAG GGC CTC ATC CCG ATG GAG GGC GAC GAG GAG 858 Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro Met Glu Gly Asp Glu Glu 120 125 130CGC TAC CTC ATC GAT AAG GGC CTC ATC CCG ATG GAG GGC GAC GAG GAG 858 Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro Met Glu Gly Asp Glu Glu 120 125 130
CTC AAG ATG CTC GCC TTC GAC ATC GAG ACG CTC TAC CAC GAG GGC GAG 906 Leu Lys Met Leu Ala Phe Asp Ile Glu Thr Leu Tyr His Glu Gly Glu 135 140 145 150CTC AAG ATG CTC GCC TTC GAC ATC GAG ACG CTC TAC CAC GAG GGC GAG 906 Leu Lys Met Leu Ala Phe Asp Ile Glu Thr Leu Tyr His Glu Gly Glu 135 140 145 150
GAG TTC GCC GAG GGG CCT ATT CTT ATG ATA AGC TAT GCC GAC GAG GAA 954 Glu Phe Ala Glu Gly Pro Ile Leu Met Ile Ser Tyr Ala Asp Glu Glu 155 160 165GAG TTC GCC GAG GGG CCT ATT CTT ATG ATA AGC TAT GCC GAC GAG GAA 954 Glu Phe Ala Glu Gly Pro Ile Leu Met Ile Ser Tyr Ala Asp Glu Glu 155 160 165
GGG GCG AGG GTA ATA ACC TGG AAG AAG ATC GAC CTT CCC TAC GTT GAC 1002 Gly Ala Arg Val Ile Thr Trp Lys Lys Ile Asp Leu Pro Tyr Val Asp 170 175 180GGG GCG AGG GTA ATA ACC TGG AAG AAG ATC GAC CTT CCC TAC GTT GAC 1002 Gly Ala Arg Val Ile Thr Trp Lys Lys Ile Asp Leu Pro Tyr Val Asp 170 175 180
GTC GTT TCA ACG GAG AAG GAG ATG ATA AAG CGC TTC CTG AAG GTT GTC 1050 Val Val Ser Thr Glu Lys Glu Met Ile Lys Arg Phe Leu Lys Val Val 185 190 195GTC GTT TCA ACG GAG AAG GAG ATG ATA AAG CGC TTC CTG AAG GTT GTC 1050 Val Val Ser Thr Glu Lys Glu Met Ile Lys Arg Phe Leu Lys Val Val 185 190 195
AAG GAG AAG GAC CCC GAT GTC CTC ATA ACC TAC AAC GGC GAC AAC TTC 1098 Lys Glu Lys Asp Pro Asp Val Leu Ile Thr Tyr Asn Gly Asp Asn Phe 200 205 210AAG GAG AAG GAC CCC GAT GTC CTC ATA ACC TAC AAC GGC GAC AAC TTC 1098 Lys Glu Lys Asp Pro Asp Val Leu Ile Thr Tyr Asn Gly Asp Asn Phe 200 205 210
GAC TTC GCT TAC CTC AAG AAG CGC TCC GAG AAG CTC GGC GTT AAG TTC 1146 Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu Lys Leu Gly Val Lys Phe 215 220 225 230GAC TTC GCT TAC CTC AAG AAG CGC TCC GAG AAG CTC GGC GTT AAG TTC 1146 Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu Lys Leu Gly Val Lys Phe 215 220 225 230
ATC CTC GGA AGG GAC GGC AGC GAG CCG AAG ATA CAG AGG ATG GGC GAC 1194 Ile Leu Gly Arg Asp Gly Ser Glu Pro Lys Ile Gin Arg Met Gly Asp 235 240 245ATC CTC GGA AGG GAC GGC AGC GAG CCG AAG ATA CAG AGG ATG GGC GAC 1194 Ile Leu Gly Arg Asp Gly Ser Glu Pro Lys Ile Gin Arg Met Gly Asp 235 240 245
CGC TTC GCC GTC GAG GTG AAG GGA AGA ATA CAC TTC GAC CTC TAC CCC 1242 Arg Phe Ala Val Glu Val Lys Gly Arg Ile His Phe Asp Leu Tyr Pro 250 255 260 GTC ATA AGA CAC ACC ATC AAC CTG CCC ACC TAC ACG CTG GAG GCC GTC 1290 Val Ile Arg His Thr Ile Asn Leu Pro Thr Tyr Thr Leu Glu Ala Val 265 270 275CGC TTC GCC GTC GAG GTG AAG GGA AGA ATA CAC TTC GAC CTC TAC CCC 1242 Arg Phe Ala Val Glu Val Lys Gly Arg Ile His Phe Asp Leu Tyr Pro 250 255 260 GTC ATA AGA CAC ACC ATC AAC CTG CCC ACC TAC ACG CTG GAG GCC GTC 1290 Val Ile Arg His Thr Ile Asn Leu Pro Thr Tyr Thr Leu Glu Ala Val 265 270 275
TAC GAG GCG ATT TTT GGG CAG CCA AAG GAG AAG GTC TAC GCT GAG GAG 1338 Tyr Glu Ala Ile Phe Gly Gin Pro Lys Glu Lys Val Tyr Ala Glu Glu 280 285 290TAC GAG GCG ATT TTT GGG CAG CCA AAG GAG AAG GTC TAC GCT GAG GAG 1338 Tyr Glu Ala Ile Phe Gly Gin Pro Lys Glu Lys Val Tyr Ala Glu Glu 280 285 290
ATA GCG CAG GCC TGG GAA ACG GGC GAG GGG CTT GAG CGC GTC GCG CGC 1386 Ile Ala Gin Ala Trp Glu Thr Gly Glu Gly Leu Glu Arg Val Ala Arg 295 300 305 310ATA GCG CAG GCC TGG GAA ACG GGC GAG GGG CTT GAG CGC GTC GCG CGC 1386 Ile Ala Gin Ala Trp Glu Thr Gly Glu Gly Leu Glu Arg Val Ala Arg 295 300 305 310
TAC TCG ATG GAG GAC GCC AAG GTA ACC TAC GAG CTG GGA AGG GAG TTC 1434 Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr Glu Leu Gly Arg Glu Phe 315 320 325TAC TCG ATG GAG GAC GCC AAG GTA ACC TAC GAG CTG GGA AGG GAG TTC 1434 Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr Glu Leu Gly Arg Glu Phe 315 320 325
TTC CCG ATG GAG GCC CAA CTT TCT CGG CTG GTC GGT CAG AGC TTC TGG 1482 Phe Pro Met Glu Ala Gin Leu Ser Arg Leu Val Gly Gin Ser Phe Trp 330 335 340TTC CCG ATG GAG GCC CAA CTT TCT CGG CTG GTC GGT CAG AGC TTC TGG 1482 Phe Pro Met Glu Ala Gin Leu Ser Arg Leu Val Gly Gin Ser Phe Trp 330 335 340
GAC GTC TCG CGC TCC AGC ACC GGC AAC CTC GTC GAG TGG TAC CTC CTC 1530 Asp Val Ser Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Tyr Leu Leu 345 350 355GAC GTC TCG CGC TCC AGC ACC GGC AAC CTC GTC GAG TGG TAC CTC CTC 1530 Asp Val Ser Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Tyr Leu Leu 345 350 355
AGG AAG GCC TAC GAG AGG AAC GAG CTG GCA CCG AAC AAG CCC TCC GGC 1578 Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro Ser Gly 360 365 370AGG AAG GCC TAC GAG AGG AAC GAG CTG GCA CCG AAC AAG CCC TCC GGC 1578 Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro Ser Gly 360 365 370
AGA GAA CTT GAG AGG CGC CGC GGG GGC TAC GCC GGC GGC TAC GTC AAG 1626 Arg Glu Leu Glu Arg Arg Arg Gly Gly Tyr Ala Gly Gly Tyr Val Lys 375 400 405 410AGA GAA CTT GAG AGG CGC CGC GGG GGC TAC GCC GGC GGC TAC GTC AAG 1626 Arg Glu Leu Glu Arg Arg Arg Gly Gly Tyr Ala Gly Gly Tyr Val Lys 375 400 405 410
GAG CCG GAG AGG GGA CTT TGG GAG AAC ATA GCT TAT TTA GAT TTT AGG 1674 Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile Ala Tyr Leu Asp Phe Arg 415 420 425GAG CCG GAG AGG GGA CTT TGG GAG AAC ATA GCT TAT TTA GAT TTT AGG 1674 Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile Ala Tyr Leu Asp Phe Arg 415 420 425
TGT CAT CCT GCC GAC ACT AAA GTC ATT GTC AAA GGG AAG GGC GTT GTA 1722 Cys His Pro Ala Asp Thr Lys Val Ile Val Lys Gly Lys Gly Val Val 430 435 440TGT CAT CCT GCC GAC ACT AAA GTC ATT GTC AAA GGG AAG GGC GTT GTA 1722 Cys His Pro Ala Asp Thr Lys Val Ile Val Lys Gly Lys Gly Val Val 430 435 440
AAC ATC AGC GAA GTT AGG GAG GGG GAC TAC GTT CTC GGC ATA GAC GGC 1770 Asn Ile Ser Glu Val Arg Glu Gly Asp Tyr Val Leu Gly Ile Asp Gly 445 450 455AAC ATC AGC GAA GTT AGG GAG GGG GAC TAC GTT CTC GGC ATA GAC GGC 1770 Asn Ile Ser Glu Val Arg Glu Gly Asp Tyr Val Leu Gly Ile Asp Gly 445 450 455
TGG CAG AAG GTT CAA AGG GTC TGG GAG TAT GAT TAC GAG GGA GAA CTC 1818 Trp Gin Lys Val Gin Arg Val Trp Glu Tyr Asp Tyr Glu Gly Glu Leu 460 465 470TGG CAG AAG GTT CAA AGG GTC TGG GAG TAT GAT TAC GAG GGA GAA CTC 1818 Trp Gin Lys Val Gin Arg Val Trp Glu Tyr Asp Tyr Glu Gly Glu Leu 460 465 470
GTA AAT ATA AAC GGC CTT AAG TGC ACA CCG AAC CAT AAG CTT CCG GTC 1866 Val Asn Ile Asn Gly Leu Lys Cys Thr Pro Asn His Lys Leu Pro Val 475 480 485 490GTA AAT ATA AAC GGC CTT AAG TGC ACA CCG AAC CAT AAG CTT CCG GTC 1866 Val Asn Ile Asn Gly Leu Lys Cys Thr Pro Asn His Lys Leu Pro Val 475 480 485 490
GTT AGG AGG ACT GAG AGG CAG ACT GCG ATA AGG GAC AGC CTT GCA AAG 1914 Val Arg Arg Thr Glu Arg Gin Thr Ala Ile Arg Asp Ser Leu Ala Lys 495 500 505GTT AGG AGG ACT GAG AGG CAG ACT GCG ATA AGG GAC AGC CTT GCA AAG 1914 Val Arg Arg Thr Glu Arg Gin Thr Ala Ile Arg Asp Ser Leu Ala Lys 495 500 505
TCT TTT CTC ACG AAA AAA GTT AAA GGT AAG CTG ATA ACC ACG CCT CTC 1962 Ser Phe Leu Thr Lys Lys Val Lys Gly Lys Leu Ile Thr Thr Pro Leu 510 515 520 TTT GAA AAA ATC GGG AAG ATC GAG CGA GAG GAC GTG CCA GAA GAG GAG 2010 Phe Glu Lys Ile Gly Lys Ile Glu Arg Glu Asp Val Pro Glu Glu Glu 525 530 535TCT TTT CTC ACG AAA AAA GTT AAA GGT AAG CTG ATA ACC ACG CCT CTC 1962 Ser Phe Leu Thr Lys Lys Val Lys Gly Lys Leu Ile Thr Thr Pro Leu 510 515 520 TTT GAA AAA ATC GGG AAG ATC GAG CGA GAG GAC GTG CCA GAA GAG GAG 2010 Phe Glu Lys Ile Gly Lys Ile Glu Arg Glu Asp Val Pro Glu Glu Glu 525 530 535
ATA CTC AAA GGA GAA CTC GCC GGA ATA ATC CTG GCT GAG GGC ACA CTC 2058 Ile Leu Lys Gly Glu Leu Ala Gly Ile Ile Leu Ala Glu Gly Thr Leu 540 545 550ATA CTC AAA GGA GAA CTC GCC GGA ATA ATC CTG GCT GAG GGC ACA CTC 2058 Ile Leu Lys Gly Glu Leu Ala Gly Ile Leu Ala Glu Gly Thr Leu 540 545 550
CTG AGA AAG GAT GTC GAG TAC TTT GAC TCT TCC AGA GGG AAG AAG AGA 2106 Leu Arg Lys Asp Val Glu Tyr Phe Asp Ser Ser Arg Gly Lys Lys Arg 555 560 565 570CTG AGA AAG GAT GTC GAG TAC TTT GAC TCT TCC AGA GGG AAG AAG AGA 2106 Leu Arg Lys Asp Val Glu Tyr Phe Asp Ser Ser Arg Gly Lys Lys Arg 555 560 565 570
GTA TCA CAC CAG TAC AGG GTT GAA ATA ACC GTT GGG GCG CAG GAG GAG 2154 Val Ser His Gin Tyr Arg Val Glu Ile Thr Val Gly Ala Gin Glu Glu 575 580 585GTA TCA CAC CAG TAC AGG GTT GAA ATA ACC GTT GGG GCG CAG GAG GAG 2154 Val Ser His Gin Tyr Arg Val Glu Ile Thr Val Gly Ala Gin Glu Glu 575 580 585
GAC TTC CAG AGG AGG ATC GTT TAC ATT TTC GAA CGC CTC TTT GGG GTA 2202 Asp Phe X Arg Arg Ile Val Tyr Ile Phe Glu Arg Leu Phe Gly Val 590 595 600GAC TTC CAG AGG AGG ATC GTT TAC ATT TTC GAA CGC CTC TTT GGG GTA 2202 Asp Phe X Arg Arg Ile Val Tyr Ile Phe Glu Arg Leu Phe Gly Val 590 595 600
ACT CCC AGT GTT TAC CGG AAA AAG AAC ACA AAC GCA ATA ACG TTC AAA 2250 Thr Pro Ser Val Tyr Arg Lys Lys Asn Thr Asn Ala Ile Thr Phe Lys 605 610 615ACT CCC AGT GTT TAC CGG AAA AAG AAC ACA AAC GCA ATA ACG TTC AAA 2250 Thr Pro Ser Val Tyr Arg Lys Lys Asn Thr Asn Ala Ile Thr Phe Lys 605 610 615
GTT GCC AAA AAA GAG GTT TAT CTT AGG GTT AGG GAA ATT ATG GAT GGC 2298 Val Ala Lys Lys Glu Val Tyr Leu Arg Val Arg Glu Ile Met Asp Gly 620 625 630GTT GCC AAA AAA GAG GTT TAT CTT AGG GTT AGG GAA ATT ATG GAT GGC 2298 Val Ala Lys Lys Glu Val Tyr Leu Arg Val Arg Glu Ile Met Asp Gly 620 625 630
ATT GAG AAC CTC CAC GCT CCT TCT GTG TTA AGG GGC TTT TTT GAA GGA 2346 Ile Glu Asn Leu His Ala Pro Ser Val Leu Arg Gly Phe Phe Glu Gly 635 640 645 650ATT GAG AAC CTC CAC GCT CCT TCT GTG TTA AGG GGC TTT TTT GAA GGA 2346 Ile Glu Asn Leu His Ala Pro Ser Val Leu Arg Gly Phe Phe Glu Gly 635 640 645 650
GAC GGA AGC GTC AAC AAG GTC CGG AAG ACA GTG GTA GTG AAT CAG GGC 2394 Asp Gly Ser Val Asn Lys Val Arg Lys Thr Val Val Val Asn Gin Gly 655 660 665GAC GGA AGC GTC AAC AAG GTC CGG AAG ACA GTG GTA GTG AAT CAG GGC 2394 Asp Gly Ser Val Asn Lys Val Arg Lys Thr Val Val Val Asn Gin Gly 655 660 665
ACC AAT AAT GAA TGG AAA ATT GAA GTG GTG TCA AAA CTC CTC AAC AAG 2442 Thr Asn Asn Glu Trp Lys Ile Glu Val Val Ser Lys Leu Leu Asn Lys 670 675 680ACC AAT AAT GAA TGG AAA ATT GAA GTG GTG TCA AAA CTC CTC AAC AAG 2442 Thr Asn Asn Glu Trp Lys Ile Glu Val Val Ser Lys Leu Leu Asn Lys 670 675 680
TTG GGG ATT CCG CAT AGA AGG TAC ACA TAC GAT TAC ACC GAA AGA GAA 2490 Leu Gly Ile Pro His Arg Arg Tyr Thr Tyr Asp Tyr Thr Glu Arg Glu 685 690 695TTG GGG ATT CCG CAT AGA AGG TAC ACA TAC GAT TAC ACC GAA AGA GAA 2490 Leu Gly Ile Pro His Arg Arg Tyr Thr Tyr Asp Tyr Thr Glu Arg Glu 685 690 695
AAA ACC ATG ACA ACG CAT ATA CTT GAG ATA GCC GGC AGG GAT GGG TTA 2538 Lys Thr Met Thr Thr His Ile Leu Glu Ile Ala Gly Arg Asp Gly Leu 700 705 710AAA ACC ATG ACA ACG CAT ATA CTT GAG ATA GCC GGC AGG GAT GGG TTA 2538 Lys Thr Met Thr Thr His Ile Leu Glu Ile Ala Gly Arg Asp Gly Leu 700 705 710
ATC CTT TTC CAG ACC ATT GTG GGA TTC ATA AGC ACT GAG AAG AAC ATG 2586 Ile Leu Phe Gin Thr Ile Val Gly Phe Ile Ser Thr Glu Lys Asn Met 715 720 725 730ATC CTT TTC CAG ACC ATT GTG GGA TTC ATA AGC ACT GAG AAG AAC ATG 2586 Ile Leu Phe Gin Thr Ile Val Gly Phe Ile Ser Thr Glu Lys Asn Met 715 720 725 730
GCG CTG GAG GAG GCA ATC AGG AAC AGG GAA GTG AAC CGC CTA GAA AAC 2634 Ala Leu Glu Glu Ala Ile Arg Asn Arg Glu Val Asn Arg Leu Glu Asn 735 740 745GCG CTG GAG GAG GCA ATC AGG AAC AGG GAA GTG AAC CGC CTA GAA AAC 2634 Ala Leu Glu Glu Ala Ile Arg Asn Arg Glu Val Asn Arg Leu Glu Asn 735 740 745
AAT GCC TTC TAT ACC CTA GCC GAC TTT ACG GCG AAG ACA GAG TAC TAC 2682 Asn Ala Phe Tyr Thr Leu Ala Asp Phe Thr Ala Lys Thr Glu Tyr Tyr 750 755 780 AAG GGC AAA GTT TAC GAC TTA ACC CTT GAG GGA ACG CCC TAT TAC TTC 2730 Lys Gly Lys Val Tyr Asp Leu Thr Leu Glu Gly Thr Pro Tyr Tyr Phe 785 790 795AAT GCC TTC TAT ACC CTA GCC GAC TTT ACG GCG AAG ACA GAG TAC TAC 2682 Asn Ala Phe Tyr Thr Leu Ala Asp Phe Thr Ala Lys Thr Glu Tyr Tyr 750 755 780 AAG GGC AAA GTT TAC GAC TTA ACC CTT GAG GGA ACG CCC TAT TAC TTC 2730 Lys Gly Lys Val Tyr Asp Leu Thr Leu Glu Gly Thr Pro Tyr Tyr Phe 785 790 795
GCC AAT GGC ATA CTG ACC CAC AAT TCG CTA TAT CCT TCG ATT ATA ATT 2778 Ala Asn Gly Ile Leu Thr His Asn Ser Leu Tyr Pro Ser Ile Ile Ile 800 805 810GCC AAT GGC ATA CTG ACC CAC AAT TCG CTA TAT CCT TCG ATT ATA ATT 2778 Ala Asn Gly Ile Leu Thr His Asn Ser Leu Tyr Pro Ser Ile Ile 800 805 810
TCC CAC AAC GTC TCC CCC GAT ACG CTC AAC CGC GAG GGC TGC GGG GAG 2826 Ser His Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Gly Glu 815 820 825 830TCC CAC AAC GTC TCC CCC GAT ACG CTC AAC CGC GAG GGC TGC GGG GAG 2826 Ser His Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Gly Glu 815 820 825 830
TAC GAC GAG GCT CCG CAG GTA GGG CAT CGC TTT TGT AAG GAC TTC CCC 2874 Tyr Asp Glu Ala Pro Gin Val Gly His Arg Phe Cys Lys Asp Phe Pro 835 840 845TAC GAC GAG GCT CCG CAG GTA GGG CAT CGC TTT TGT AAG GAC TTC CCC 2874 Tyr Asp Glu Ala Pro Gin Val Gly His Arg Phe Cys Lys Asp Phe Pro 835 840 845
GGC TTC ATC CCC AGC CTC CTC GGT GAC CTG CTC GAC GAG AGG CAG AAG 2922 Gly Phe Ile Pro Ser Leu Leu Gly Asp Leu Leu Asp Glu Arg Gin Lys 855 860 865GGC TTC ATC CCC AGC CTC CTC GGT GAC CTG CTC GAC GAG AGG CAG AAG 2922 Gly Phe Ile Pro Ser Leu Leu Gly Asp Leu Leu Asp Glu Arg Gin Lys 855 860 865
GTA AAG AAG CAC ATG AAG GCC ACG GTG GAC CCG ATA GAG AAG AAG CTC 2970 Val Lys Lys His Met Lys Ala Thr Val Asp Pro Ile Glu Lys Lys Leu 870 875 880GTA AAG AAG CAC ATG AAG GCC ACG GTG GAC CCG ATA GAG AAG AAG CTC 2970 Val Lys Lys His Met Lys Ala Thr Val Asp Pro Ile Glu Lys Lys Leu 870 875 880
CTC GAT TAC AGG CAG CGC GCA ATT AAA ATC CTC GCC AAC AGC TTC TAC 3018 Leu Asp Tyr Arg Gin Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr 885 890 895CTC GAT TAC AGG CAG CGC GCA ATT AAA ATC CTC GCC AAC AGC TTC TAC 3018 Leu Asp Tyr Arg Gin Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr 885 890 895
GGC TAC TAT GGC TAC GCA AAG GCC CGC TGG TAC TGC AAG GAG TGC GCC 3066 Gly Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala 900 905 910 915GGC TAC TAT GGC TAC GCA AAG GCC CGC TGG TAC TGC AAG GAG TGC GCC 3066 Gly Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala 900 905 910 915
GAG AGC GTT ACC GCC TGG GGC AGG CAG TAC ATT GAG ACC ACC ATG AGG 3114 Glu Ser Val Thr Ala Trp Gly Arg Gin Tyr Ile Glu Thr Thr Met Arg 920 925 930GAG AGC GTT ACC GCC TGG GGC AGG CAG TAC ATT GAG ACC ACC ATG AGG 3114 Glu Ser Val Thr Ala Trp Gly Arg Gin Tyr Ile Glu Thr Thr Met Arg 920 925 930
GAA ATA GAG GAA AAA TTT GGC TTT AAA GTG CTG TAC GCG GAT AGT GTT 3162 Glu Ile Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Ser Val 935 940 945GAA ATA GAG GAA AAA TTT GGC TTT AAA GTG CTG TAC GCG GAT AGT GTT 3162 Glu Ile Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Ser Val 935 940 945
ACA GGG GAC ACA GAG GTA ACC ATC AGA AGA AAC GGC AGG ATT GAG TTC 3210 Thr Gly Asp Thr Glu Val Thr Ile Arg Arg Asn Gly Arg Ile Glu Phe 950 955 960ACA GGG GAC ACA GAG GTA ACC ATC AGA AGA AAC GGC AGG ATT GAG TTC 3210 Thr Gly Asp Thr Glu Val Thr Ile Arg Arg Asn Gly Arg Ile Glu Phe 950 955 960
GTT CCA ATC GAG AAA CTC TTT GAG CGC GTT GAT CAC CGT GTT GGT GAG 3258 Val Pro Ile Glu Lys Leu Phe Glu Arg Val Asp His Arg Val Gly Glu 965 970 975GTT CCA ATC GAG AAA CTC TTT GAG CGC GTT GAT CAC CGT GTT GGT GAG 3258 Val Pro Ile Glu Lys Leu Phe Glu Arg Val Asp His Arg Val Gly Glu 965 970 975
AAG GAG TAC TGC GTT CTT GGA GGG GTT GAG GCA CTG ACA CTC GAC AAC 3306 Lys Glu Tyr Cys Val Leu Gly Gly Val Glu Ala Leu Thr Leu Asp Asn 980 985 990 995AAG GAG TAC TGC GTT CTT GGA GGG GTT GAG GCA CTG ACA CTC GAC AAC 3306 Lys Glu Tyr Cys Val Leu Gly Gly Val Glu Ala Leu Thr Leu Asp Asn 980 985 990 995
AGG GGC AGG CTC GTG TGG AAG AAG GTT CCG TAC GTC ATG AGA CAT AAA 3354 Arg Gly Arg Leu Val Trp Lys Lys Val Pro Tyr Val Met Arg His Lys 1000 1005 1010AGG GGC AGG CTC GTG TGG AAG AAG GTT CCG TAC GTC ATG AGA CAT AAA 3354 Arg Gly Arg Leu Val Trp Lys Lys Val Pro Tyr Val Met Arg His Lys 1000 1005 1010
ACG GAC AAA AGA ATC TAT AGG GTA TGG TTC ACC AAC TCT TGG TAC CTT 3402 Thr Asp Lys Arg Ile Tyr Arg Val Trp Phe Thr Asn Ser Trp Tyr Leu 1015 1020 1025 GAC GTG ACG GAG GAT CAC TCG CTA ATA GGC TAC CTG AAC ACA AGC AAA 3450 Asp Val Thr Glu Asp His Ser Leu Ile Gly Tyr Leu Asn Thr Ser Lys 1030 1035 1040ACG GAC AAA AGA ATC TAT AGG GTA TGG TTC ACC AAC TCT TGG TAC CTT 3402 Thr Asp Lys Arg Ile Tyr Arg Val Trp Phe Thr Asn Ser Trp Tyr Leu 1015 1020 1025 GAC GTG ACG GAG GAT CAC TCG CTA ATA GGC TAC CTG AAC ACA AGC AAA 3450 Asp Val Thr Glu Asp His Ser Leu Ile Gly Tyr Leu Asn Thr Ser Lys 1030 1035 1040
GTC AAA CCC GGA AAG CCC TTG AAA GAG CGT CTC GTC GAG GTC AAG CCA 3498 Val Lys Pro Gly Lys Pro Leu Lys Glu Arg Leu Val Glu Val Lys Pro 1045 1050 1055GTC AAA CCC GGA AAG CCC TTG AAA GAG CGT CTC GTC GAG GTC AAG CCA 3498 Val Lys Pro Gly Lys Pro Leu Lys Glu Arg Leu Val Glu Val Lys Pro 1045 1050 1055
GAA GAA TTG GGG GGT AAG GTC AAG TCT CTC ATT ACG CCC AAT CGG CCA 3546 Glu Glu Leu Gly Gly Lys Val Lys Ser Leu Ile Thr Pro Asn Arg Pro 1060 1065 1070 1075GAA GAA TTG GGG GGT AAG GTC AAG TCT CTC ATT ACG CCC AAT CGG CCA 3546 Glu Glu Leu Gly Gly Lys Val Lys Ser Leu Ile Thr Pro Asn Arg Pro 1060 1065 1070 1075
ATT GCC CGT ACC ATC AAG GCC AAC CCC ATT GCC GTC AAG CTC TGG GAG 3594 Ile Ala Arg Thr Ile Lys Ala Asn Pro Ile Ala Val Lys Leu Trp Glu 1080 1085 1090ATT GCC CGT ACC ATC AAG GCC AAC CCC ATT GCC GTC AAG CTC TGG GAG 3594 Ile Ala Arg Thr Ile Lys Ala Asn Pro Ile Ala Val Lys Leu Trp Glu 1080 1085 1090
TTA ATT GGC CTG CTG GTG GGA GAT GGC AAC TGG GGT GGA CAA TCG AAC 3642 Leu Ile Gly Leu Leu Val Gly Asp Gly Asn Trp Gly Gly Gin Ser Asn 1095 1100 1105TTA ATT GGC CTG CTG GTG GGA GAT GGC AAC TGG GGT GGA CAA TCG AAC 3642 Leu Ile Gly Leu Leu Val Gly Asp Gly Asn Trp Gly Gly Gin Ser Asn 1095 1100 1105
TGG GCC AAA TAC TAC GTT GGC CTC TCC TGT GGG CTG GAT AAA GCC GAA 3690 Trp Ala Lys Tyr Tyr Val Gly Leu Ser Cys Gly Leu Asp Lys Ala Glu 1110 1115 1120TGG GCC AAA TAC TAC GTT GGC CTC TCC TGT GGG CTG GAT AAA GCC GAA 3690 Trp Ala Lys Tyr Tyr Val Gly Leu Ser Cys Gly Leu Asp Lys Ala Glu 1110 1115 1120
ATA GAG AGA AAA GTC CTG AAC CCT TTA AGA GAG GCA AGC GTC ATC TCC 3738 Ile Glu Arg Lys Val Leu Asn Pro Leu Arg Glu Ala Ser Val Ile Ser 1125 1130 1135ATA GAG AGA AAA GTC CTG AAC CCT TTA AGA GAG GCA AGC GTC ATC TCC 3738 Ile Glu Arg Lys Val Leu Asn Pro Leu Arg Glu Ala Ser Val Ile Ser 1125 1130 1135
AAC TAC TAC GAC AAG AGC AAG AAG GGC GAC GTT TCC ATA CTC TCC AAG 3786 Asn Tyr Tyr Asp Lys Ser Lys Lys Gly Asp Val Ser Ile Leu Ser Lys 1140 1145 1150 1155AAC TAC TAC GAC AAG AGC AAG AAG GGC GAC GTT TCC ATA CTC TCC AAG 3786 Asn Tyr Tyr Asp Lys Ser Lys Lys Gly Asp Val Ser Ile Leu Ser Lys 1140 1145 1150 1155
TGG CTC GCC GGA TTC ATG GTC AAA TAC TTC AAA GAT GAA AAT GGG AAC 3834 Trp Leu Ala Gly Phe Met Val Lys Tyr Phe Lys Asp Glu Asn Gly Asn 1160 1165 1170TGG CTC GCC GGA TTC ATG GTC AAA TAC TTC AAA GAT GAA AAT GGG AAC 3834 Trp Leu Ala Gly Phe Met Val Lys Tyr Phe Lys Asp Glu Asn Gly Asn 1160 1165 1170
AAG GCC ATT CCC AGC TTC ATG TTC AAC CTT CCA AGG GAA TAC ATA GAG 3882 Lys Ala Ile Pro Ser Phe Met Phe Asn Leu Pro Arg Glu Tyr Ile Glu 1175 1180 1185AAG GCC ATT CCC AGC TTC ATG TTC AAC CTT CCA AGG GAA TAC ATA GAG 3882 Lys Ala Ile Pro Ser Phe Met Phe Asn Leu Pro Arg Glu Tyr Ile Glu 1175 1180 1185
GCC TTT CTA CGG GGG CTG TTT TCA GCG GAC GGA ACG GTA AGC TTG CGT 3930 Ala Phe Leu Arg Gly Leu Phe Ser Ala Asp Gly Thr Val Ser Leu Arg 1190 1195 1200GCC TTT CTA CGG GGG CTG TTT TCA GCG GAC GGA ACG GTA AGC TTG CGT 3930 Ala Phe Leu Arg Gly Leu Phe Ser Ala Asp Gly Thr Val Ser Leu Arg 1190 1195 1200
AGA GGA ATC CCA GAA ATT AGA CTG ACA AGC GTT AAC AGA GAG CTT AGT 3978 Arg Gly Ile Pro Glu Ile Arg Leu Thr Ser Val Asn Arg Glu Leu Ser 1205 1210 1215AGA GGA ATC CCA GAA ATT AGA CTG ACA AGC GTT AAC AGA GAG CTT AGT 3978 Arg Gly Ile Pro Glu Ile Arg Leu Thr Ser Val Asn Arg Glu Leu Ser 1205 1210 1215
GAT GCC GTG AGA AAG TTG CTG TGG CTG GTT GGG GTC TCC AAC TCA CTA 4026 Asp Ala Val Arg Lys Leu Leu Trp Leu Val Gly Val Ser Asn Ser Leu 1220 1225 1230 1235GAT GCC GTG AGA AAG TTG CTG TGG CTG GTT GGG GTC TCC AAC TCA CTA 4026 Asp Ala Val Arg Lys Leu Leu Trp Leu Val Gly Val Ser Asn Ser Leu 1220 1225 1230 1235
TTC ACC GAA ACC AAG CCA AAC CGG TAC CTG GAG AAA GAA AGT GGA ACG 4074 Phe Thr Glu Thr Lys Pro Asn Arg Tyr Leu Glu Lys Glu Ser Gly Thr 1240 1245 1250TTC ACC GAA ACC AAG CCA AAC CGG TAC CTG GAG AAA GAA AGT GGA ACG 4074 Phe Thr Glu Thr Lys Pro Asn Arg Tyr Leu Glu Lys Glu Ser Gly Thr 1240 1245 1250
CAT TCG ATT CAC GTG AGG ATA AAG AAC AAG CAT CGC TTT GCC GAT AGA 4122 His Ser Ile His Val Arg Ile Lys Asn Lys His Arg Phe Ala Asp Arg 1255 1260 1265 ATA GGC TTT CTC ATA GAC AGA AAA TCC ACC AAA CTC TCC GAG AAC CTG 4170 Ile Gly Phe Leu Ile Asp Arg Lys Ser Thr Lys Leu Ser Glu Asn Leu 1270 1275 1280CAT TCG ATT CAC GTG AGG ATA AAG AAC AAG CAT CGC TTT GCC GAT AGA 4122 His Ser Ile His Val Arg Ile Lys Asn Lys His Arg Phe Ala Asp Arg 1255 1260 1265 ATA GGC TTT CTC ATA GAC AGA AAA TCC ACC AAA CTC TCC GAG AAC CTG 4170 Ile Gly Phe Leu Ile Asp Arg Lys Ser Thr Lys Leu Ser Glu Asn Leu 1270 1275 1280
GGG GGA CAT ACA AAC AAG AAG AGG GCT TAC AAA TAT GAT TTT GAC TTG 4218 Gly Gly His Thr Asn Lys Lys Arg Ala Tyr Lys Tyr Asp Phe Asp Leu 1285 1290 1295GGG GGA CAT ACA AAC AAG AAG AGG GCT TAC AAA TAT GAT TTT GAC TTG 4218 Gly Gly His Thr Asn Lys Lys Arg Ala Tyr Lys Tyr Asp Phe Asp Leu 1285 1290 1295
GTA TAC CCC AGA AAA ATC GAA GAG ATA ACC TAC GAC GGC TAC GTC TAT 4266 Val Tyr Pro Arg Lys Ile Glu Glu Ile Thr Tyr Asp Gly Tyr Val Tyr 1300 1305 1310 1315GTA TAC CCC AGA AAA ATC GAA GAG ATA ACC TAC GAC GGC TAC GTC TAT 4266 Val Tyr Pro Arg Lys Ile Glu Glu Ile Thr Tyr Asp Gly Tyr Val Tyr 1300 1305 1310 1315
GAC ATC GAG GTT GAG GGA ACC CAC AGG TTC TTC GCC AAC GGA ATA CTC 4314 Asp Ile Glu Val Glu Gly Thr His Arg Phe Phe Ala Asn Gly Ile Leu 1320 1325 1330GAC ATC GAG GTT GAG GGA ACC CAC AGG TTC TTC GCC AAC GGA ATA CTC 4314 Asp Ile Glu Val Glu Gly Thr His Arg Phe Phe Ala Asn Gly Ile Leu 1320 1325 1330
GTT CAC AAC ACA GAC GGC TTT TTC GCA ACA ATC CCC GGA GCG GAC GCC 4362 Val His Asn Thr Asp Gly Phe Phe Ala Thr Ile Pro Gly Ala Asp Ala 1335 1340 1345GTT CAC AAC ACA GAC GGC TTT TTC GCA ACA ATC CCC GGA GCG GAC GCC 4362 Val His Asn Thr Asp Gly Phe Phe Ala Thr Ile Pro Gly Ala Asp Ala 1335 1340 1345
GAG ACG GTC AAA AAG AAG GCC AGG GAG TTC CTT AAC TAC ATT AAC CCC 4410 Glu Thr Val Lys Lys Lys Ala Arg Glu Phe Leu Asn Tyr Ile Asn Pro 1350 1355 1360GAG ACG GTC AAA AAG AAG GCC AGG GAG TTC CTT AAC TAC ATT AAC CCC 4410 Glu Thr Val Lys Lys Lys Ala Arg Glu Phe Leu Asn Tyr Ile Asn Pro 1350 1355 1360
AAG CTG CCC GGT CTC CTC GAA CTC GAG TAC GAG GGC TTC TAC AGG CGC 4458 Lys Leu Pro Gly Leu Leu Glu Leu Glu Tyr Glu Gly Phe Tyr Arg Arg 1365 1370 1375AAG CTG CCC GGT CTC CTC GAA CTC GAG TAC GAG GGC TTC TAC AGG CGC 4458 Lys Leu Pro Gly Leu Leu Glu Leu Glu Tyr Glu Gly Phe Tyr Arg Arg 1365 1370 1375
GGT TTC TTC GTA ACC AAG AAG AAG TAC GCG GTG ATA GAC GAG GAG GGC 4506 Gly Phe Phe Val Thr Lys Lys Lys Tyr Ala Val Ile Asp Glu Glu Gly 1380 1385 1390 1395GGT TTC TTC GTA ACC AAG AAG AAG TAC GCG GTG ATA GAC GAG GAG GGC 4506 Gly Phe Phe Val Thr Lys Lys Lys Tyr Ala Val Ile Asp Glu Glu Gly 1380 1385 1390 1395
AAG ATA ACG ACG CGC GGG CTT GAG ATC GTC CGG CGC GAC TGG AGT GAG 4554 Lys Ile Thr Thr Arg Gly Leu Glu Ile Val Arg Arg Asp Trp Ser Glu 1400 1405 1410AAG ATA ACG ACG CGC GGG CTT GAG ATC GTC CGG CGC GAC TGG AGT GAG 4554 Lys Ile Thr Thr Arg Gly Leu Glu Ile Val Arg Arg Asp Trp Ser Glu 1400 1405 1410
GTG GCT AAG GAG ACG CAG GCG AGG GTC TTG GAG GCC ATA CTG CGG CAC 4602 Val Ala Lys Glu Thr Gin Ala Arg Val Leu Glu Ala Ile Leu Arg His 1415 1420 1425GTG GCT AAG GAG ACG CAG GCG AGG GTC TTG GAG GCC ATA CTG CGG CAC 4602 Val Ala Lys Glu Thr Gin Ala Arg Val Leu Glu Ala Ile Leu Arg His 1415 1420 1425
GGT GAC GTC GAG GAG GCC GTG AGG ATT GTC AAG GAA GTG ACG GAA AAG 4650 Gly Asp Val Glu Glu Ala Val Arg Ile Val Lys Glu Val Thr Glu Lys 1430 1435 1440GGT GAC GTC GAG GAG GCC GTG AGG ATT GTC AAG GAA GTG ACG GAA AAG 4650 Gly Asp Val Glu Glu Ala Val Arg Ile Val Lys Glu Val Thr Glu Lys 1430 1435 1440
CTG AGC AAG TAC GAG GTT CCG CCA GAG AAG CTC GTC ATC CAC GAG CAG 4698 Leu Ser Lys Tyr Glu Val Pro Pro Glu Lys Leu Val Ile His Glu Gin 1445 1450 1455CTG AGC AAG TAC GAG GTT CCG CCA GAG AAG CTC GTC ATC CAC GAG CAG 4698 Leu Ser Lys Tyr Glu Val Pro Pro Glu Lys Leu Val Ile His Glu Gin 1445 1450 1455
ATT ACC AGG GAG CTG AAG GAC TAC AAG GCC ACC GGC CCG CAC GTG GCC 4746 Ile Thr Arg Glu Leu Lys Asp Tyr Lys Ala Thr Gly Pro His Val Ala 1460 1465 1470 1475ATT ACC AGG GAG CTG AAG GAC TAC AAG GCC ACC GGC CCG CAC GTG GCC 4746 Ile Thr Arg Glu Leu Lys Asp Tyr Lys Ala Thr Gly Pro His Val Ala 1460 1465 1470 1475
ATA GCG AAG CGC CTC GCC GCG AGG GGG ATT AAG GTT CGC CCT GGG ACA 4794 Ile Ala Lys Arg Leu Ala Ala Arg Gly Ile Lys Val Arg Pro Gly Thr 1480 1485 1490ATA GCG AAG CGC CTC GCC GCG AGG GGG ATT AAG GTT CGC CCT GGG ACA 4794 Ile Ala Lys Arg Leu Ala Ala Arg Gly Ile Lys Val Arg Pro Gly Thr 1480 1485 1490
GTC ATC AGC TAC ATC GTC CTG AAA GGT TCC GGC AGG ATA GGG GAC AGG 4842 Val Ile Ser Tyr Ile Val Leu Lys Gly Ser Gly Arg Ile Gly Asp Arg 1495 1500 1505 ACG ATA CCC TTC GAC GAG TTC GAC CCC ACG AAG CAC AGG TAC GAT GCG 4890 Thr Ile Pro Phe Asp Glu Phe Asp Pro Thr Lys His Arg Tyr Asp Ala 1510 1515 1520GTC ATC AGC TAC ATC GTC CTG AAA GGT TCC GGC AGG ATA GGG GAC AGG 4842 Val Ile Ser Tyr Ile Val Leu Lys Gly Ser Gly Arg Ile Gly Asp Arg 1495 1500 1505 ACG ATA CCC TTC GAC GAG TTC GAC CCC ACG AAG CAC AGG TAC GAT GCG 4890 Thr Ile Pro Phe Asp Glu Phe Asp Pro Thr Lys His Arg Tyr Asp Ala 1510 1515 1520
GAG TAC TAC ATC GAG AAC CAG GTT CTG CCG GCG GTG GAG AGA ATC CTC 4938 Glu Tyr Tyr Ile Glu Asn Gin Val Leu Pro Ala Val Glu Arg Ile Leu 1525 1530 1535GAG TAC TAC ATC GAG AAC CAG GTT CTG CCG GCG GTG GAG AGA ATC CTC 4938 Glu Tyr Tyr Ile Glu Asn Gin Val Leu Pro Ala Val Glu Arg Ile Leu 1525 1530 1535
AAG GCC TTC GGC TAC AAG AAG GAG GAT TTG CGC TAC CAG AAG ACG CGG 4986 Lys Ala Phe Gly Tyr Lys Lys Glu Asp Leu Arg Tyr Gin Lys Thr Arg 1540 1545 1550 1555AAG GCC TTC GGC TAC AAG AAG GAG GAT TTG CGC TAC CAG AAG ACG CGG 4986 Lys Ala Phe Gly Tyr Lys Lys Glu Asp Leu Arg Tyr Gin Lys Thr Arg 1540 1545 1550 1555
CAG GTT GGG CTG GGG GCG TGG CTC AAA ATG GGG AAG AAA TGA 5028CAG GTT GGG CTG GGG GCG TGG CTC AAA ATG GGG AAG AAA TGA 5028
Gin Val Gly Leu Gly Ala Trp Leu Lys Met Gly Lys LysGin Val Gly Leu Gly Ala Trp Leu Lys Met Gly Lys Lys
1560 1565 15681560 1565 1568
AGGCCAAGCT T 5039AGGCCAAGCT T 5039
(2) INFORMATION POUR LA SEQ ID NO : 2 :(2) INFORMATION FOR SEQ ID NO: 2:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 774 acides aminés (ii) TYPE DE MOLECULE: protéine (ix) CARACTERISTIQUES(A) LENGTH: 774 amino acids (ii) TYPE OF MOLECULE: protein (ix) CHARACTERISTICS
(A) NOM/CLE: ADN polymerase de THERMOCOCCUS fumicolans Tfu (xi) DESCRIPTION DE LA SEQUENCES: SEQ ID NO : 2 :(A) NAME / KEY: DNA polymerase of THERMOCOCCUS fumicolans Tfu (xi) DESCRIPTION OF THE SEQUENCES: SEQ ID NO: 2:
Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Arg Pro Val Ile 1 5 10 15Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Arg Pro Val Ile 1 5 10 15
Arg Val Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30Arg Val Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30
Asp Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45Asp Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45
Glu Asp Val Lys Lys Ile Thr Ala Ser Arg His Gly Thr Thr Val Arg 50 55 60Glu Asp Val Lys Lys Ile Thr Ala Ser Arg His Gly Thr Thr Val Arg 50 55 60
Val Val Arg Ala Gly Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80Val Val Arg Ala Gly Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gin Asp Val Pro Ala Ile 85 90 95Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gin Asp Val Pro Ala Ile 85 90 95
Arg Asp Lys Ile Arg Glu His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110Arg Asp Lys Ile Arg Glu His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125
Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155 160Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155 160
Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190
Arg Phe Leu Lys Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205Arg Phe Leu Lys Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu 210 215 220Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu 210 215 220
Lys Leu Gly Val Lys Phe Ile Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240Lys Leu Gly Val Lys Phe Ile Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240
Ile Gin Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255Ile Gin Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255
His Phe Asp Leu Tyr Pro Val Ile Arg His Thr Ile Asn Leu Pro Thr 260 265 270His Phe Asp Leu Tyr Pro Val Ile Arg His Thr Ile Asn Leu Pro Thr 260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Gin Pro Lys Glu 275 280 285Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Gin Pro Lys Glu 275 280 285
Lys Val Tyr Ala Glu Glu Ile Ala Gin Ala Trp Glu Thr Gly Glu Gly 290 295 300Lys Val Tyr Ala Glu Glu Ile Ala Gin Ala Trp Glu Thr Gly Glu Gly 290 295 300
Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320
Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gin Leu Ser Arg Leu 325 330 335Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gin Leu Ser Arg Leu 325 330 335
Val Gly Gin Ser Phe Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350Val Gly Gin Ser Phe Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350
Val Glu Trp Tyr Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365Val Glu Trp Tyr Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365
Pro Asn Lys Pro Ser Gly Arg Glu Leu Glu Arg Arg Arg Gly Gly Tyr 370 375 380Pro Asn Lys Pro Ser Gly Arg Glu Leu Glu Arg Arg Arg Gly Gly Tyr 370 375 380
Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile 385 390 395 400Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile 385 390 395 400
Ala Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Ser His 405 405 410Ala Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Ser His 405 405 410
Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Gly Glu Tyr Asp 415 420 425Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Gly Glu Tyr Asp 415 420 425
Glu Ala Pro GÏn Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe 430 435 440Glu Ala Pro GÏn Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe 430 435 440
Ile Pro Ser Leu Leu Gly Asp Leu Leu Asp Glu Arg Gin Lys Val Lys 445 450 455Ile Pro Ser Leu Leu Gly Asp Leu Leu Asp Glu Arg Gin Lys Val Lys 445 450 455
Lys His Met Lys Ala Thr Val Asp Pro Ile Glu Lys Lys Leu Leu Asp 460 465 470 475Lys His Met Lys Ala Thr Val Asp Pro Ile Glu Lys Lys Leu Leu Asp 460 465 470 475
Tyr Arg Gin Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr 480 485 490 Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 495 500 505Tyr Arg Gin Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr 480 485 490 Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 495 500 505
Val Thr Ala Trp Gly Arg Gin Tyr Ile Glu Thr Thr Met Arg Glu Ile 510 515 520Val Thr Ala Trp Gly Arg Gin Tyr Ile Glu Thr Thr Met Arg Glu Ile 510 515 520
Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 525 530 535Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 525 530 535
Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 540 545 555 560Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 540 545 555 560
Arg Glu Phe Leu Asn Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu 565 570 575Arg Glu Phe Leu Asn Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu 565 570 575
Leu Glu Tyr Glu Gly Phe Tyr Arg Arg Gly Phe Phe Val Thr Lys Lys 580 585 590Leu Glu Tyr Glu Gly Phe Tyr Arg Arg Gly Phe Phe Val Thr Lys Lys 580 585 590
Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605
Glu Ile Val Arg Arg Asp Trp Ser Glu Val Ala Lys Glu Thr Gin Ala 610 615 620Glu Ile Val Arg Arg Asp Trp Ser Glu Val Ala Lys Glu Thr Gin Ala 610 615 620
Arg Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu Glu Ala Val 625 630 635 640Arg Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu Glu Ala Val 625 630 635 640
Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655
Pro Glu Lys Leu Val Ile His Glu Gin Ile Thr Arg Glu Leu Lys Asp 660 665 670Pro Glu Lys Leu Val Ile His Glu Gin Ile Thr Arg Glu Leu Lys Asp 660 665 670
Tyr Lys Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Ala 675 680 685Tyr Lys Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Ala 675 680 685
Arg Gly Ile Lys Val Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700Arg Gly Ile Lys Val Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700
Lys Gly Ser Gly Arg Ile Gly Asp Arg Thr Ile Pro Phe Asp Glu Phe 705 710 715 720Lys Gly Ser Gly Arg Ile Gly Asp Arg Thr Ile Pro Phe Asp Glu Phe 705 710 715 720
Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gin 725 730 735Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gin 725 730 735
Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Lys Lys 740 745 750Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Lys Lys 740 745 750
Glu Asp Leu Arg Tyr Gin Lys Thr Arg Gin Val Gly Leu Gly Ala Trp 755 760 765Glu Asp Leu Arg Tyr Gin Lys Thr Arg Gin Val Gly Leu Gly Ala Trp 755 760 765
Leu Lys Met Gly Lys Lys 770 774 (2) INFORMATION POUR LA SEQ ID NO : 3 :Leu Lys Met Gly Lys Lys 770 774 (2) INFORMATION FOR SEQ ID NO: 3:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 360 acides aminés (ii) TYPE DE MOLECULE: protéine ( ix ) CARACTERISTIQUES(A) LENGTH: 360 amino acids (ii) TYPE OF MOLECULE: protein (ix) CHARACTERISTICS
(A) NOM/CLE: intéine I-Tfu-1 (xi) DESCRIPTION DE LA SEQUENCES: SEQ ID NO: 3 :(A) NAME / KEY: intein I-Tfu-1 (xi) DESCRIPTION OF THE SEQUENCES: SEQ ID NO: 3:
Cys His Pro Ala Asp Thr Lys Val Ile Val Lys Gly Lys Gly Val Val 1 5 10 15Cys His Pro Ala Asp Thr Lys Val Ile Val Lys Gly Lys Gly Val Val 1 5 10 15
Asn Ile Ser Glu Val Arg Glu Gly Asp Tyr Val Leu Gly Ile Asp Gly 20 25 30Asn Ile Ser Glu Val Arg Glu Gly Asp Tyr Val Leu Gly Ile Asp Gly 20 25 30
Trp Gin Lys Val Gin Arg Val Trp Glu Tyr Asp Tyr Glu Gly Glu Leu 35 40 45Trp Gin Lys Val Gin Arg Val Trp Glu Tyr Asp Tyr Glu Gly Glu Leu 35 40 45
Val Asn Ile Asn Gly Leu Lys Cys Thr Pro Asn His Lys Leu Pro Val 50 55 60Val Asn Ile Asn Gly Leu Lys Cys Thr Pro Asn His Lys Leu Pro Val 50 55 60
Val Arg Arg Thr Glu Arg Gin Thr Ala Ile Arg Asp Ser Leu Ala Lys 65 70 75 80Val Arg Arg Thr Glu Arg Gin Thr Ala Ile Arg Asp Ser Leu Ala Lys 65 70 75 80
Ser Phe Leu Thr Lys Lys Val Lys Gly Lys Leu Ile Thr Thr Pro Leu 85 90 95Ser Phe Leu Thr Lys Lys Val Lys Gly Lys Leu Ile Thr Thr Pro Leu 85 90 95
Phe Glu Lys Ile Gly Lys Ile Glu Arg Glu Asp Val Pro Glu Glu Glu 100 105 110Phe Glu Lys Ile Gly Lys Ile Glu Arg Glu Asp Val Pro Glu Glu Glu 100 105 110
Ile Leu Lys Gly Glu Leu Ala Gly Ile Ile Leu Ala Glu Gly Thr Leu 115 120 125Ile Leu Lys Gly Glu Leu Ala Gly Ile Leu Ala Glu Gly Thr Leu 115 120 125
Leu Arg Lys Asp Val Glu Tyr Phe Asp Ser Ser Arg Gly Lys Lys Arg 130 135 140Leu Arg Lys Asp Val Glu Tyr Phe Asp Ser Ser Arg Gly Lys Lys Arg 130 135 140
Val Ser His Gin Tyr Arg Val Glu Ile Thr Val Gly Ala Gin Glu Glu 145 150 155 160Val Ser His Gin Tyr Arg Val Glu Ile Thr Val Gly Ala Gin Glu Glu 145 150 155 160
Asp Phe X Arg Arg Ile Val Tyr Ile Phe Glu Arg Leu Phe Gly Val 165 170 175Asp Phe X Arg Arg Ile Val Tyr Ile Phe Glu Arg Leu Phe Gly Val 165 170 175
Thr Pro Ser Val Tyr Arg Lys Lys Asn Thr Asn Ala Ile Thr Phe Lys 180 185 190Thr Pro Ser Val Tyr Arg Lys Lys Asn Thr Asn Ala Ile Thr Phe Lys 180 185 190
Val Ala Lys Lys Glu Val Tyr Leu Arg Val Arg Glu Ile Met Asp Gly 195 200 205Val Ala Lys Lys Glu Val Tyr Leu Arg Val Arg Glu Ile Met Asp Gly 195 200 205
Ile Glu Asn Leu His Ala Pro Ser Val Leu Arg Gly Phe Phe Glu Gly 210 215 220Ile Glu Asn Leu His Ala Pro Ser Val Leu Arg Gly Phe Phe Glu Gly 210 215 220
Asp Gly Ser Val Asn Lys Val Arg Lys Thr Val Val Val Asn Gin Gly 225 230 235 240Asp Gly Ser Val Asn Lys Val Arg Lys Thr Val Val Val Asn Gin Gly 225 230 235 240
Thr Asn Asn Glu Trp Lys Ile Glu Val Val Ser Lys Leu Leu Asn Lys 245 250 255Thr Asn Asn Glu Trp Lys Ile Glu Val Val Ser Lys Leu Leu Asn Lys 245 250 255
Leu Gly Ile Pro His Arg Arg Tyr Thr Tyr Asp Tyr Thr Glu Arg Glu 260 265 270 Lys Thr Met Thr Thr His I le Leu Glu I le Ala Gly Arg Asp Gly Leu 275 280 285Leu Gly Ile Pro His Arg Arg Tyr Thr Tyr Asp Tyr Thr Glu Arg Glu 260 265 270 Lys Thr Met Thr Thr His I Leu Glu I Ala Gly Arg Asp Gly Leu 275 280 285
Ile Leu Phe Gin Thr Ile Val Gly Phe Ile Ser Thr Glu Lys Asn Met 290 295 300Ile Leu Phe Gin Thr Ile Val Gly Phe Ile Ser Thr Glu Lys Asn Met 290 295 300
Ala Leu Glu Glu Ala Ile Arg Asn Arg Glu Val Asn Arg Leu Glu Asn 305 310 315 320Ala Leu Glu Glu Ala Ile Arg Asn Arg Glu Val Asn Arg Leu Glu Asn 305 310 315 320
Asn Ala Phe Tyr Thr Leu Ala Asp Phe Thr Ala Lys Thr Glu Tyr Tyr 325 330 335Asn Ala Phe Tyr Thr Leu Ala Asp Phe Thr Ala Lys Thr Glu Tyr Tyr 325 330 335
Lys Gly Lys Val Tyr Asp Leu Thr Leu Glu Gly Thr Pro Tyr Tyr Phe 340 345 350Lys Gly Lys Val Tyr Asp Leu Thr Leu Glu Gly Thr Pro Tyr Tyr Phe 340 345 350
Ala Asn Gly Ile Leu Thr His Asn 355 360Ala Asn Gly Ile Leu Thr His Asn 355 360
(2 ) INFORMATION POUR LA SEQ ID NO : 4 :(2) INFORMATION FOR SEQ ID NO: 4:
( i ) CARACTRERI STIQUES DE LA SEQUENCE :(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 389 acides aminés ( ii ) TYPE DE MOLECULE : protéine ( ix ) CARACTERI STIQUES(A) LENGTH: 389 amino acids (ii) TYPE OF MOLECULE: protein (ix) CHARACTERISTICS
(A) NOM/CLE : intéine I-Tfu-2 (xi ) DESCRIPTION DE LA SEQUENCES : SEQ ID NO : 4 :(A) NAME / KEY: intein I-Tfu-2 (xi) DESCRIPTION OF THE SEQUENCES: SEQ ID NO: 4:
Ser Val Thr Gly Asp Thr Glu Val Thr I le Arg Arg Asn Gly Arg I le 1 5 10 15Ser Val Thr Gly Asp Thr Glu Val Thr I le Arg Arg Asn Gly Arg I le 1 5 10 15
Glu Phe Val Pro Ile Glu Lys Leu Phe Glu Arg Val Asp His Arg Val 20 25 30Glu Phe Val Pro Ile Glu Lys Leu Phe Glu Arg Val Asp His Arg Val 20 25 30
Gly Glu Lys Glu Tyr Cys Val Leu Gly Gly Val Glu Ala Leu Thr Leu 35 40 45Gly Glu Lys Glu Tyr Cys Val Leu Gly Gly Val Glu Ala Leu Thr Leu 35 40 45
Asp Asn Arg Gly Arg Leu Val Trp Lys Lys Val Pro Tyr Val Met Arg 50 55 60Asp Asn Arg Gly Arg Leu Val Trp Lys Lys Val Pro Tyr Val Met Arg 50 55 60
His Lys Thr Asp Lys Arg I le Tyr Arg Val Trp Phe Thr Asn Ser Trp 65 70 75 80His Lys Thr Asp Lys Arg I le Tyr Arg Val Trp Phe Thr Asn Ser Trp 65 70 75 80
Tyr Leu Asp Val Thr Glu Asp His Ser Leu Ile Gly Tyr Leu Asn Thr 85 90 95Tyr Leu Asp Val Thr Glu Asp His Ser Leu Ile Gly Tyr Leu Asn Thr 85 90 95
Ser Lys Val Lys Pro Gly Lys Pro Leu Lys Glu Arg Leu Val Glu Val 100 105 110Ser Lys Val Lys Pro Gly Lys Pro Leu Lys Glu Arg Leu Val Glu Val 100 105 110
Lys Pro Glu Glu Leu Gly Gly Lys Val Lys Ser Leu Ile Thr Pro Asn 115 120 125Lys Pro Glu Glu Leu Gly Gly Lys Val Lys Ser Leu Ile Thr Pro Asn 115 120 125
Arg Pro Ile Ala Arg Thr Ile Lys Ala Asn Pro Ile Ala Val Lys Leu 130 135 140Arg Pro Ile Ala Arg Thr Ile Lys Ala Asn Pro Ile Ala Val Lys Leu 130 135 140
Trp Glu Leu Ile Gly Leu Leu Val Gly Asp Gly Asn Trp Gly Gly Gin 145 150 155 160Trp Glu Leu Ile Gly Leu Leu Val Gly Asp Gly Asn Trp Gly Gly Gin 145 150 155 160
Ser Asn Trp Ala Lys Tyr Tyr Val Gly Leu Ser Cys Gly Leu Asp Lys 165 170 175 Ala Glu Ile Glu Arg Lys Val Leu Asn Pro Leu Arg Glu Ala Ser Val 180 185 190Ser Asn Trp Ala Lys Tyr Tyr Val Gly Leu Ser Cys Gly Leu Asp Lys 165 170 175 Ala Glu Ile Glu Arg Lys Val Leu Asn Pro Leu Arg Glu Ala Ser Val 180 185 190
Ile Ser Asn Tyr Tyr Asp Lys Ser Lys Lys Gly Asp Val Ser Ile Leu 195 200 205Ile Ser Asn Tyr Tyr Asp Lys Ser Lys Lys Gly Asp Val Ser Ile Leu 195 200 205
Ser Lys Trp Leu Ala Gly Phe Met Val Lys Tyr Phe Lys Asp Glu Asn 210 215 220Ser Lys Trp Leu Ala Gly Phe Met Val Lys Tyr Phe Lys Asp Glu Asn 210 215 220
Gly Asn Lys Ala Ile Pro Ser Phe Met Phe Asn Leu Pro Arg Glu Tyr 225 230 235 240Gly Asn Lys Ala Ile Pro Ser Phe Met Phe Asn Leu Pro Arg Glu Tyr 225 230 235 240
Ile Glu Ala Phe Leu Arg Gly Leu Phe Ser Ala Asp Gly Thr Val Ser 245 250 255Ile Glu Ala Phe Leu Arg Gly Leu Phe Ser Ala Asp Gly Thr Val Ser 245 250 255
Leu Arg Arg Gly Ile Pro Glu Ile Arg Leu Thr Ser Val Asn Arg Glu 260 265 270Leu Arg Arg Gly Ile Pro Glu Ile Arg Leu Thr Ser Val Asn Arg Glu 260 265 270
Leu Ser Asp Ala Val Arg Lys Leu Leu Trp Leu Val Gly Val Ser Asn 275 280 285Leu Ser Asp Ala Val Arg Lys Leu Leu Trp Leu Val Gly Val Ser Asn 275 280 285
Ser Leu Phe Thr Glu Thr Lys Pro Asn Arg Tyr Leu Glu Lys Glu Ser 290 295 300Ser Leu Phe Thr Glu Thr Lys Pro Asn Arg Tyr Leu Glu Lys Glu Ser 290 295 300
Gly Thr His Ser Ile His Val Arg Ile Lys Asn Lys His Arg Phe Ala 305 310 315 320Gly Thr His Ser Ile His Val Arg Ile Lys Asn Lys His Arg Phe Ala 305 310 315 320
Asp Arg Ile Gly Phe Leu Ile Asp Arg Lys Ser Thr Lys Leu Ser Glu 325 330 335Asp Arg Ile Gly Phe Leu Ile Asp Arg Lys Ser Thr Lys Leu Ser Glu 325 330 335
Asn Leu Gly Gly His Thr Asn Lys Lys Arg Ala Tyr Lys Tyr Asp Phe 340 345 350Asn Leu Gly Gly His Thr Asn Lys Lys Arg Ala Tyr Lys Tyr Asp Phe 340 345 350
Asp Leu Val Tyr Pro Arg Lys Ile Glu Glu Ile Thr Tyr Asp Gly Tyr 355 360 365Asp Leu Val Tyr Pro Arg Lys Ile Glu Glu Ile Thr Tyr Asp Gly Tyr 355 360 365
Val Tyr Asp Ile Glu Val Glu Gly Thr His Arg Phe Phe Ala Asn Gly 370 375 380Val Tyr Asp Ile Glu Val Glu Gly Thr His Arg Phe Phe Ala Asn Gly 370 375 380
Ile Leu Val His Asn 385 389 Ile Leu Val His Asn 385 389

Claims

REVENDICATIONS
1) ADN polymerase purifiée thermostable d ' archabactéries de l'espèce Thermococcus fumicolans ayant un poids moléculaire de l'ordre de 89 000 daltons et ses dérivés enzymatiquement équivalents .1) purified thermostable DNA polymerase of archabacteria of the species Thermococcus fumicolans having a molecular weight of the order of 89,000 daltons and its enzymatically equivalent derivatives.
2 ) ADN polymerase selon la revendication 1 dont la séquence en acides aminés est représentée dans la liste de séquence en annexe sous le numéro SEQ ID NO:l ou un fragment de celle-ci ou encore un assemblage de tels fragments .2) DNA polymerase according to claim 1, the amino acid sequence of which is represented in the annexed sequence list under the number SEQ ID NO: 1 or a fragment thereof or an assembly of such fragments.
3 ) ADN polymerase selon la revendication 2 dont la séquence en acides aminés est représentée dans la liste de séquence en annexe sous le numéro SEQ ID NO:2.3) DNA polymerase according to claim 2, the amino acid sequence of which is represented in the annexed sequence list under the number SEQ ID NO: 2.
4) Une séquence d'ADN constituée par ou comprenant la séquence codant pour une ADN polymerase selon quelconque des revendications 1 à 3.4) A DNA sequence constituted by or comprising the sequence coding for a DNA polymerase according to any one of claims 1 to 3.
5) Une séquence d'ADN selon la revendication 4 constituée par ou comprenant la séquence comprise entre les nucleotides 357 à 5028 de la séquence SED ID NO : 1, ou un fragment de celle-ci ou encore un assemblage de tels fragments .5) A DNA sequence according to claim 4 consisting of or comprising the sequence between nucleotides 357 to 5028 of the sequence SED ID NO: 1, or a fragment thereof or an assembly of such fragments.
6) Une séquence d'ADN selon l'une des revendications 4 à 5 constituée par ou comprenant les nucleotides 357 à 1674 et 2755 à 3156 et 4324 à 5028 de la séquence d'ADN représentée dans la liste de séquences en annexe sous le numéro SED ID NO : 1.6) A DNA sequence according to one of claims 4 to 5 consisting of or comprising nucleotides 357 to 1674 and 2755 to 3156 and 4324 to 5028 of the DNA sequence represented in the sequence list in the annex under the number SED ID NO: 1.
7) Un vecteur contenant la séquence d'ADN de l'une quelconque des revendications 4 à 6. 8) Un hôte transformé par un vecteur selon la revendication 7.7) A vector containing the DNA sequence of any one of claims 4 to 6. 8) A host transformed by a vector according to claim 7.
9) Procédé de préparation d'une ADN polymerase thermostable d'archaebactéries de l'espèce Thermococcus fumicolans , caractérisé en ce que l'on cultive l'hôte selon la revendication 8 dans des conditions permettant 1 ' expression de ladite ADN polymerase et en ce que 1 ' on extrait et récupère celle-ci par tout moyen approprié.9) Process for the preparation of a thermostable DNA polymerase of archaebacteria of the species Thermococcus fumicolans, characterized in that the host is cultivated according to claim 8 under conditions allowing the expression of said DNA polymerase and in that 1 extract and recover it by any suitable means.
10) Procédé d'amplification enzymatique d'une séquence d'acide nucléique caractérisé en ce que l'on met en oeuvre une ADN polymerase thermostable selon l'une quelconque des revendications 1 à 3.10) Method of enzymatic amplification of a nucleic acid sequence characterized in that a thermostable DNA polymerase according to any one of claims 1 to 3 is used.
11) Intéine purifiée thermostable d'archaebactéries de l'espèce Thermococcus fumicolans.11) Purified thermostable intein from archaebacteria of the species Thermococcus fumicolans.
FEUILLE RECTIFIEE (REGLE 91) ISA/EP RECTIFIED SHEET (RULE 91) ISA / EP
PCT/FR1997/000761 1997-04-29 1997-04-29 Thermostable dna polymerase and inteins of the thermococcus fumicolans species WO1998049274A1 (en)

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PCT/FR1998/000868 WO1998049275A2 (en) 1997-04-29 1998-04-29 Archaebacterium intein of the thermococcus fumicolans species

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US8637288B2 (en) 2000-02-17 2014-01-28 Qiagen, Gmbh Thermostable chimeric nucleic acid polymerases and uses thereof
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