Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Patent
1993-02-03
1995-05-30
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
935 10, 935 14, 536 232, 536 234, C12N 1500, C12N 912
Patent
active
054200296
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a purified, thermostable DNA polymerase purified from the hyperthermophilic eubactefia Thermotoga maritima and means for isolating and producing the enzyme. Thermostable DNA polymerases are useful in many recombinant DNA techniques, especially nucleic acid amplification by the polymerase chain reaction (PCR).
BACKGROUND ART
In Huber et al., 1986, Arch. Microbiol. 144:324-333, the isolation of the bacterium Thermotoga maritima is described. T. maritima is a eubacterium that is strictly anaerobic, rod-shaped, fermentative, hyperthermophilic, and grows between 55.degree. C. and 90.degree. C., with an optimum growth temperature of about 80.degree. C. This eubacterium has been isolated from geothermally heated sea floors in Italy and the Azores. T. maritima cells have a sheath-like structure and monotrichous flagellation. T. maritima is classified in the eubacterial kingdom by virtue of having murein and fatty acid-containing lipids, diphtheria-toxin-resistant elongation factor 2, an RNA polymerase subunit pattern, and sensitivity to antibiotics.
Extensive research has been conducted on the isolation of DNA polymerases from mesophilic microorganisms such as E. coli. See, for example, Bessman et al., 1957, i. Bio. Chem. 223:171-177, and Buttin and Kornberg, 1966, J. Biol. Chem. 241:5419-5427. Much less investigation has been made on the isolation and purification of DNA polymerases from thermophiles such as Thermotoga maritima. In Kaledin et al., 1980, Biokhymiya 45:644-651, a six-step isolation and enrichment procedure for DNA polymerase activity from cells of a Thermus aquaticus YT-1 strain is disclosed. These steps involve isolation of crude extract, DEAE-cellulose chromatography, fractionation on hydroxyapatite, fractionation on DEAE-cellulose, and chromatography on single-strand DNA-cellulose. The molecular weight of the purified enzyme is reported by Kaledin et al. as 62,000 daltons per monomeric unit.
A second enrichment scheme for a polymerase from Thermus aquaticus is described in Chien et al., 1976, J. Bacteriol. 127:1550-1557. In this process, the crude extract is applied to a DEAE-Sephadex column. The dialyzed pooled fractions are then subjected to treatment on a phosphocellulose column. The pooled fractions are dialyzed, and bovine serum albumin (BSA) is added to prevent loss of polymerase activity. The resulting mixture is loaded on a DNA-cellulose column. The pooled material from the column is dialyzed. The molecular weight of the purified protein is reported to be about 63,000 daltons to 68,000 daltons.
The use of thermostable enzymes, such as those described in Chien et al. and Kaledin et al., to amplify existing nucleic acid sequences in amounts that are large compared to the amount initially present is described in U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188, which describe the PCR process, each of which is incorporated herein by reference. Primers, template, nucleoside triphosphates, the appropriate buffer and reaction conditions, and polymerase are used in the PCR process, which involves denaturation of target DNA, hybridization of primers, and synthesis of complementary strands. The extension product of each primer becomes a template for the production of the desired nucleic acid sequence. The patents disclose that, if the polymerase employed is a thermostable enzyme, then polymerase need not be added after every denaturation step, because heat will not destroy the polymerase activity.
U.S. Pat. No. 4,889,818, European Patent Publication No. 258,017, and PCT Publication No. 89/06691, the disclosures of which are incorporated herein by reference, describe the isolation and recombinant expression of an .about.94 kDa thermostable DNA polymerase from Thermus aquaticus and the use of that polymerase in PCR. Although T. aquaticus DNA polymerase is especially preferred for use in PCR and other recombinant DNA techniques, there remains a need for other thermostable polymerases.
Accordingly, there is a desire in the art to produce a purifie
REFERENCES:
patent: 4889818 (1989-12-01), Gelfand et al.
patent: 4965188 (1990-10-01), Mullis et al.
Simpson et al., 1990, "Purification and Some Properties of a Thermostable DNA Polymerase From a Thermotoga Species" Biochem. Cell Biol. 68:1292-1296.
Simpson et al., 1990, "Purification of a Thermostable DNA Polymerase From a Thermotoga Species" Ann. New York Acad. Sci. 613:426-428.
Lawyer et al., 1989, "Isolation, Characterization, and Expression in Escherichia coli of the DNA Polymerase Gene From Thermus Aquaticus" J. Biol. Chem. 264(11):6427-6437.
Matsuzawa et al., 1988, "Purification and Characterization of Aqualysin I (a thermophilic alkaline serine protease) Produced by Thermus Aquaticus YT-1" Eur. J. Biochem. 171:441-447.
Ruttimann et al., 1985, "DNA Polymerases From the Extremely Thermophilic Bacterium Thermus Thermophilus HB-8" Eur. J. Biochem 149:41-46.
Gelfand et al., 1989, "Taq DNA Polymerase" PCR Technology, Principles and Applications for DNA Amplification, Chapter 2, pp. 17-22 (1989), Ed. by H. A. Erlich.
Leavitt and Ito, 1989, "T5 DNA Polymerase: Structural-Functional Relationships to Other DNA Polymerases" Proc. Natl. Acad. Sci. USA 86:4465-4469.
Bernad et al., 1989, "A Conserved 3'-5' Exonuclease Active Site in Prokaryotic and Eukaryotic DNA Polymerases" Cell 59:219-228.
Huber et al., 1986, "Thermotoga Maritima sp. nov. Represents a New Genus of Unique Extremely Thermophilic Eubacteria Growing Up to 90" Chem. Abstracts 105:386 (Abstract No. 38901u).
Chien et al., 1976, "Deoxyribonucleic Acid Polymerase From the Extreme Thermophile Thermus Aquaticus" J. Bacteriology 127(3):1550-1557.
Suggs et al., 1981, "Use of Synthetic Oligonucleotides as Hybridization Probes: Isolation of Cloned cDNA Sequences for Human B2-Microglobulin" Proc. Natl. Acad. Sci. USA 78(11):6613-6617.
Young and Davis, 1983, "Efficient Isolation of Genes by Using Antibody Probes" Proc. Natl. Acad. Sci. USA 80:1194-1198.
Kaledin et al., 1980, "Isolation and Properties of DNA Polymerase From Extremely Thermophilic Bacterium Thermus Aquatics YT1" Biokhimiya 45(4):644-651.
Barany et al., 1991, "The Ligase Chain Reaction in a PCR World" PCR Methods and Applications 1(1):5-16.
Simpson et al., 1990, "Purification and Some Properties of a Thermostable DNA Polymerase From a Thermotoga Species" Biochem. Cell Biol. 68:1292-1296.
Simpson et al., 1990, "Purification of a Thermostable DNA Polymerase From a Thermotoga Species" Ann. New York Acad. Sci. 613:426-428.
Gelfand David H.
Lawyer Frances C.
Stoffel Susanne
Gould George M.
Hendricks Keith D.
Hoffmann-La Roche Inc.
Sias Stacey R.
Tramaloni Dennis P.
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