DE544789T1 - Gereinigtes thermostabiles nukleinsäure-polymeraseenzym aus thermotoga maritima. - Google Patents

Gereinigtes thermostabiles nukleinsäure-polymeraseenzym aus thermotoga maritima.

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Publication number
DE544789T1
DE544789T1 DE91915802T DE91915802T DE544789T1 DE 544789 T1 DE544789 T1 DE 544789T1 DE 91915802 T DE91915802 T DE 91915802T DE 91915802 T DE91915802 T DE 91915802T DE 544789 T1 DE544789 T1 DE 544789T1
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enzyme
dna sequence
enzyme according
extract
recombinant
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David Gelfand
Frances Lawyer
Susanne Stoffel
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F Hoffmann La Roche AG
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F Hoffmann La Roche AG
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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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/10—Nucleotidyl transfering
    • C12Q2521/101—DNA polymerase

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  • Life Sciences & Earth Sciences (AREA)
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  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
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  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
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  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Saccharide Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Claims (24)

VOSSlUS & FARTNER PATEfJTANVVALT-- EP 91 91 5802.2 s££ÄS§?4 ' 5' ^ »93 £ÄS§4 F. Hoffmann-La Roche AG &Lgr;TELEFON 474q75 u.Z.: EP-2573 Patentansprüche
1. Ein gereinigtes thermostabiles DNA Polymerase-I-Enzym, das die Verknüpfung von Nukleosidtriphosphaten katalysiert, um damit einen Nukleinsäurestrang komplementär zu einem Nukleinsäure-Matrizenstrang zu bilden, wobei das Enzym aus dem Eubakterium Thermotoga maritima herstammt.
2. Das Enzym gemäss Anspruch 1, das ein Molekulargewicht zwischen ungefähr 97 Kilodaltons bis 103 Kilodaltons hat.
3. Das Enzym gemäss Anspruch 1, welches reversible Transcriptase-Aktivität besitzt.
4. Das Enzym gemäss Anspruch 1, welches 3'—»5'-Exonuclease-Aktivität besitzt.
5. Das Enzym gemäss Anspruch 1 in natürlicher Form.
6. Das Enzym gemäss Anspruch 2 in natürlicher Form.
]5 7. Das Enzym gemäss Anspruch 1 in rekombinanter Form.
8. Das Enzym gemäss Anspruch 2 in rekombinanter Form.
9. Das Enzym gemäss Anspruch 2, das reversible Transcriptase- und 3'-»5'-Exonuclease-Aktivität besitzt.
10. Das Enzym gemäss Anspruch 9 in natürlicher Form.
11. Das Enzym gemäss Anspruch 9 in rekombinanter Form.
12. Ein Verfahren zur Reinigung von Thermotoga maritima-DNA Polymerase aus T. maritima-Zellen. wobei das Verfahren folgende Schritte umfasst:
(a) Herstellung eines rohen Zellextraktes aus obengenannten Zellen;
(b) Anpassung der Ionenstärke dieses Extraktes, so dass diese Polymerase sich von jeglicher Nukleinsäure in diesem Extrakt löst;
(c) der Extrakt einer hydrophoben Wechselwirkungschromatographie unterworfen wird;
(d) der Extrakt einer DNA-Bindeprotein-AffinitätschromatogTaphie
unterworfen wird;
(e) der Extrakt einer Nukleotidbindeprotein-Affinitätschromatographie unterworfen wird; und
(f) der Extrakt einer Chromatographie ausgewählt aus der Gruppe
bestehend aus Anionenaustausch-Chromatographie, Kationenaustausch-Chromatographie und Hydroxyapatit-Chromatographie unterworfen wird.
13. Eine rekombinante DNA-Sequenz, welche Thermotoga maritima-DNA-Polymerase I-Aktivität kodiert.
14. Die DNA-Sequenz gemäss Anspruch 13, welche die Aminosäuresequenz von SEQ ID NO: 1, vom Amino- bis zum Carboxyterminus, kodiert:
1 MARLFLFDGT ALAYRAYYAV DRSLSTSTGI PTNATYDVAR MLVRFIKDHI
51 IVGKDYVAVA FDKKAATFRH KLLITYKAQR PKTPDLLIQQ LPYIKKLVZA
101 LGMKVLEVEG YEADDIIATL AVKGLPLFDE IFIVTGDKDM LQLVNEKIKV
151 WRIVKGISDL ELYDAQKVKE KYGVEPQQIP DLLALTGDEI DNIPGVTGIG
201 EKTAVQLLEK YKDLEDILNH VRELPQKVRK ALLRDRENAI LSKKLAILZT
251 NVPIEINWEE LRYQGYDREK LLPLLKELEF ASIMKELQLY EESEPVGYRI
301 VKDLVEFEKL lEKLRESPSF AIDLETSSLD PFDCDIVGIS VSFKPKEAiY
351 IPLHHRNAQN LDEKEVLKKL KEILEDPGAK IVAQNLKFDY KVLMVKGVE?
4 01 VPPYFDTMIA AYLLEPNEKK FNLDDLALKF LGYKMTSYQE LMSFSFPLFG
4 51 FSFADVPVEK AANYSCEDAD ITYRLYKTLS LKLHEADLEN VFYKIEMPLV
501 NVLARMELNG VYVDTEFLKK LSEEYGKKLE ELAEEIYRIA GEFFNINSPK
551 QVSRILFEKL GIKPRGKTTK TGDYSTRIEV LEELAGEHEI IPLILEYRKI
601 QKLKSTYIDA LPKMVNPKTG RIHA5FNQTG TATGRLSSSD PNLENLPTr.S
651 EEGKEIRKAI VPQDPNWWIV SADYSQIELR ILAHLSGDEN LLP-AFEEGID
701 VHTLTASRIF NVKPEEVTEE MRRAGKMVNF SIIYGVTPYG LSVRLGVrVK
751 EAEKMIVNYF VLYPKVRDYI QRWSEAKEK GYVRTLFGRK RDIPQLMA=D
801 RNTQAEGERI AINTPIQGTA ADIIKLAMIE IDRELKERKM RSKMIIQVHD
851 ELVFEVPNEE KDALVELVKD RMTNWKLSV PLEVDVTIGK TWS
15. Die DNA-Sequenz gemäss Anspruch 14, welche die DNA-Sequenz von SEQID NO: list:
1 ATGGCGAGAC TATTTCTCTT TGATGGAACT GCTCTGGCCT ACAGAGCGTA
51 CTATGCGGTC GATAGATCGC TTTCTACTTC CACCGGCATT CCCACAAACG 101 CCACATACGA TGTGGCGAGG ATGCTGGTGA GATTCATCAA AGACCATATC
151 ATTGTCGGAA AAGACTACGT AGACTTACAA TTCGACAAAA CCAAAGACTC 201 CTTCAGACAC AAGCTCCTCG CTTCCGTACA GGCTCAAAGA GGTCGAAGCC 251 CGGATCTCCT GATTCAGCAG GGTAGAAGGA TAAAGAAGCT ACGATATAAT 301 CTTGGAATGA AAGTGCTGGA GGCTTCCGCT TACGAAGCGG ATATTCATAG 351 TGCCACTCTG GCTGTGAAGG CTTCAGCTTG TTTTGATGAA GATCAAGGTG 401 TGACCGGAGA TAAAGACATG ATCCGATCTG TGAACGAAAA ATGCGCAGAA 451 TGGCGAATCG TAAAAGGGAT TTGAACCCCA GAACTTTACG GATCTTCTGG 501 GGTGAAGGAA AAATACGGTG GACAACATCC GCAGATCCCG TGGGATAGGT 551 CTCTAACCGG AGATGAAATA TCTAGAGAAG CCGGTGTAAC TCGAAGACAT 601 GAAAAGACTG CTGTTCAGCT TTCCTCAAAA TACAAAGACC GCCCTGCTTC 651 ACTGAATCAT GTTCGCGAAC CTCAGCAAAA GGTGAGAAAA TCTGGAAACA 701 GAGACAGAGA AAACGCCATT CTGGGAAGAA AGCTGGCGAT AGGGCTACGA 751 AACGTTCCCA TTGAAATAAA TTTTGAAAGA CTTCGCTACC GCATCCATCA 801 CAGAGAGAAA CTCTTACCAC GAAGAGTCCG ACTGGAATTC ATACAGAATA 851 TGAAGGAACT TCAACTGTAC TGAAAAACTC AACCCGTTGG TGAGAGAATC 901 GTGAAAGACC TAGTGGAATT TTGAGACGTC ATAGAGAAAC CCTTTCGACT 951 CCCTTCGTTC GCCATAGATC GTGTCTTTCA TTCCCTCGAT AGCGTACTAC 1001 GCGACATTGT CGGTATCTCT CGCCCAGAAC AACCAAAGGA AAGAGGTTCT 1051 ATACCACTCC ATCATAGAAA TGGAGGACCC CTGGACGAAA ATCGTTGCTC 1101 GAAAAAGCTC AAAGAAATTC AAGGTGTTGA CGGAGCAAAG TGTTGAACCT 1151 AGAATTTGAA ATTCGATTAC GATGATAGCG TGGTGAAGGG TTGAGCCGAA 1201 GTTCCTCCTT ACTTCGACAC ACGATCTCGC GCTTACCTTC CTTGGATACA 1251 CGAAAAGAAG TTCAATCTGG CTCATGTCCT ATTGAAATTT GCTGTTTGGT 1301 AAATGACATC TTACCAAGAG TGTAGAAAAA TCTCTTTTCC ACTCCTGTGA 1351 TTCAGTTTTG CCGATGTTCC GACTTTACAA GCAGCGAACT TTAAAACTCC 1401 AGATGCAGAC ATCACCTACA GTGTTCTACA GACCCTGAGC GCCCCTTGTG 1451 ACGAGGCAGA TCTGGAAAAC ACTGAACGGT AGATAGAAAT ACACAGAGTT 1501 AACGTGCTTG CACGGATGGA AGTACGGAAA GTGTATGTGG GAACTGGCAG 1551 CCTGAAGAAA CTCTCAGAAG GGAGAGCCGT AAAACTCGAA CTCACCGAAG 1601 AGGAAATATA CAGGATAGCT TGAAAAACTC TCAACATAAA CACGTGGTAA 1651 CAGGTTTCAA GGATCCTTTT ATTCAACACG GGCATAAAAC CTCGAGGAAC 1701 AACGACGAAA ACGGGAGACT ATTCCTCTGA CATAGAAGTC CAGAAAGATA 1751 TTGCCGGTGA ACACGAAATC CATAGACGCT TTCTTGAATA TGGTCAACCC 1801 CAGAAATTGA AATCAACCTA CTTCTTTCAA CTTCCCAAGA ACTGCCACTG 1851 AAAGACCGGA AGGATTCATG CCCAATCTTG TCAAACGGGG GACGAAAAGT 1901 GAAGACTTAG CAGCAGCGAT GAAAGCGATA AGAACCTCCC ATCCAAACTG 1951 GAAGAGGGAA AAGAAATCAG ACTCCCAAAT GTTCCTCAGG ATCCTCGCCC 2001 GTGGATCGTC AGTGCCGACT CTTTTGAGGG AGAACTGAGG GGGCATCGAC 2051 ATCTCAGTGG TGATGAGAAT CATTCGAAGA
-A-
2101 GTCCACACTC TAACAGCTTC CAGAATATTC AACGTGAAAC CCGAAGAAGT
2151 AACCGAAGAA ATGCGCCGCG CTGGTAAAAT GGTTAATTTT TCCATCATAT
2201 ACGGTGTAAC ACCTTACGGT CTGTCTGTGA GGCTTGGAGT ACCTGTGAAA
2251 GAAGCAGAAA AGATGATCGT CAACTACTTC GTCCTCTACC CAAAGGTGCG
2301 CGATTACATT CAGAGGGTCG TATCGGAAGC GAAAGAAAAA GGCTATGTTA
2351 GAACGCTGTT TGGAAGAAAA AGAGACATAC CACAGCTCAT GGCCCCGGAC
2401 AGGAACACAC AGGCTGAAGG AGAACGAATT GCCATAAACA CTCCCATACA
2451 GGGTACAGCA GCGGATATAA TAAAGCTGGC TATGATAGAA ATAGACAGGG
2501 AACTGAAAGA AAGAAAAATG AGATCGAAGA TGATCATACA GGTCCACGAC
2551 GAACTGGTTT TTGAAGTGCC CAATGAGGAA AAGGACGCGC TCGTCGAGCT
2 601 GGTGAAAGAC AGAATGACGA ATGTGGTAAA GCTTTCAGTG CCGCTCGAAG
2 651 TGGATGTAAC CATCGGCAAA ACATGGTCGT GA
16. Ein rekombinanter DNA-Vektor, welcher die DNA-Sequenz gemäss Anspruch 13 enthält.
17. Eine rekombinante Wirtszelle, die mit dem Vektor gemäss Anspruch 16 transformiert ist.
18. Eine rekombinante Wirtszelle gemäss Anspruch 17, welche E.coli ist.
19. Das Enzym gemäss Anspruch 1 mit einem Molekulargewicht von etwa 86 Kilodaltons, welches in rekombinanter Form vorliegt.
20. Das Enzym gemäss Anspruch 1 mit einem Molekulargewicht von etwa 70 Kilodaltons, welches in rekombinanter Form vorliegt.
21. Die DNA-Sequenz gemäss Anspruch 13, welche die Aminosäuren von Nummer 140 bis 893 der SEQ ID NO: 1 kodiert.
22. Die DNA-Sequenz gemäss Anspruch 21, welche die Nukleotide von
Nummer 418 bis 2682 der SEQ ID NO: 1 ist.
23. Die DNA-Sequenz gemäss Anspruch 13, welche die Aminosäuren von Nummer 284 bis 893 der SEQ ID NO: 1 kodiert.
24. Die DNA-Sequenz gemäss Anspruch 23, welche die Nukleotide von Nummer 850 bis 2682 der SEQ ID NO: 1 ist.
DE91915802T 1990-08-13 1991-08-13 Gereinigtes thermostabiles nukleinsäure-polymeraseenzym aus thermotoga maritima. Pending DE544789T1 (de)

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US07/567,244 US5374553A (en) 1986-08-22 1990-08-13 DNA encoding a thermostable nucleic acid polymerase enzyme from thermotoga maritima

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DE91915802T Pending DE544789T1 (de) 1990-08-13 1991-08-13 Gereinigtes thermostabiles nukleinsäure-polymeraseenzym aus thermotoga maritima.

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US (3) US5374553A (de)
EP (1) EP0544789B1 (de)
JP (3) JPH07108220B2 (de)
AT (1) ATE233816T1 (de)
AU (1) AU653747B2 (de)
DE (2) DE69133204T2 (de)
ES (1) ES2193131T3 (de)
WO (1) WO1992003556A1 (de)

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