Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-26
2002-09-03
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S091500, C435S194000, C536S023200, C530S350000
Reexamination Certificate
active
06444424
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a substantially pure thermostable DNA polymerase. Specifically, the DNA polymerase of the present invention is a
Thermotoga neapolitana
DNA polymerase having a molecular weight of about 100 kilodaltons. The present invention also relates to cloning and expression of the
Thermotoga neapolitana
DNA polymerase in
E. coli
, to DNA molecules containing the cloned gene, and to hosts which express said genes. The DNA polymerase of the present invention may be used in DNA sequencing and amplification reactions.
2. Background Information
DNA polymerases synthesize the formation of DNA molecules which are complementary to a DNA template. Upon hybridization of a primer to the single-stranded DNA template, polymerases synthesize DNA in the 5′ to 3′ direction, successively adding nucleotides to the 3′-hydroxyl group of the growing strand. Thus, in the presence of deoxyribonucleoside triphosphates (dNTPs) and a primer, a new DNA molecule, complementary to the single stranded DNA template, can be synthesized.
A number of DNA polymerases have been isolated from mesophilic microorganisms such as
E. coli
. A number of these mesophilic DNA polymerases have also been cloned. Lin et al. cloned and expressed T4 DNA polymerase in
E. coli
(
Proc. Natl. Acad. Sci. USA
84:7000-7004 (1987)). Tabor et al. (U.S. Pat. No. 4,795,699) describes a cloned T7 DNA polymerase, while Minkley et al. (
J. Biol. Chem
. 259:10386-10392 (1984)) and Chatterjee (U.S. Pat. No. 5,047,342) described
E. coli
DNA polymerase I and cloning of T5 DNA polymerase, respectively.
Although DNA polymerases from thermophiles are known, relatively little investigation has been done to isolate and even clone these enzymes. Chien et al.,
J. Bacteriol
. 127:1550-1557 (1976) describe a purification scheme for obtaining a polymerase from
Thermus aquaticus
. The resulting protein had a molecular weight of about 63,000 daltons by gel filtration analysis and 68,000 daltons by sucrose gradient centrifugation. Kaledin et al., Biokhymiya 45:644-51 (1980) disclosed a purification procedure for isolating DNA polymerase from
T. aquaticus
YET1 strain. The purified enzyme was reported to be a 62,000 dalton monomeric protein. Gelfand et al. (U.S. Pat. No. 4,889,818) cloned a gene encoding a thermostable DNA polymerase from
Thermus aquaticus
. The molecular weight of this protein was found to be about 86,000 to 90,000 daltons.
Simpson et al. purified and partially characterized a thermostable DNA polymerase from a Thermotoga species (
Biochem. Cell. Biol
. 86:1292-1296 (1990)). The purified DNA polymerase isolated by Simpson et al. exhibited a molecular weight of 85,000 daltons as determined by SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. The enzyme exhibited half-lives of 3 minutes at 95° C. and 60 minutes at 50° C. in the absence of substrate and its pH optimum was in the range of pH 7.5 to 8.0. Triton X-100 appeared to enhance the thermostability of this enzyme. The strain used to obtain the thermostable DNA polymerase described by Simpson et al. was Thermotoga species strain FjSS3-B.1 (Hussar et al.,
FEMS Microbiology Letters
37:121-127 (1986)). Other DNA polymerases have been isolated from thermophilic bacteria including
Bacillus steraothermophilus
(Stenesh et al.,
Biochim. iochys. Acta
272:156-166 (1972); and Kaboev et al.,
J. Bacteriol
. 145:21-26 (1981)) and several archaetsipecies (Rossi et al.,
System. Appl. Microbiol
. 7:337-341 (196); Klimczak et al., Biochemistry 25:48504855 (1986); and Elie et al.,
Eur. J. Biochem
. 178:619-626 (1989)). The most extensively purified archaebacterial DNA polymerase had a reported half-life of 15 minutes at 87° C. (Elie et al. (1989), supra). Innis et al., In
PCR Protocol: A Guide To Methods and Amplification
, Academic Press, Inc., San Diego (1990) noted that there are several extreme thermophilic eubacteria and archaebacteria that are capable of growth at very high temperatures (Bergquist et al.,
Biotech. Genet. Eng. Rev
. 5:199-244 (1987); and Kelly et al.,
Biotechnol Prog
. 4:47-62 (1988)) and suggested that these organisms may contain very thermostable DNA polymerases.
SUMMARY OF THE INVENTION
The present invention is directed to a thermostable DNA polymerase having a molecular weight of about 100 kilodaltons. More specifically, the DNA polymerase of the invention is isolated from Thernotoga neapolitana (Tne). The Thernotoga species preferred for isolating the DNA polymerase of the present invention was isolated from an African continental solfataric spring (Windberger et al.,
Arch. Microbiol
. 151. 506-512, (1989)).
The Tne DNA polymerase of the present invention is extremely thermostable, showing more than 50% of activity after being heated for 60 minutes at 90° C. with or without detergent. Thus, the DNA polymerase of the present invention is more thermostable than Taq DNA polymerase.
The present invention is also directed to cloning a gene encoding a
Thermotoga neapolitana
DNA polymerase enzyme. DNA molecules containing the Tne DNA polymerase gene, according to the present invention, can be transformed and expressed in a host cell to produce a Tne DNA polymerase having a molecular weight of 100 kilodaltons. Any number of hosts may be used to express the Thermotoga DNA polymerase gene of the present invention; including prokaryotic and eukaryotic cells. Preferably, prokaryotic cells are used to express the DNA polymerase of the invention. The preferred prokaryotic hosts according to the present invention is
E. coli.
The Tne DNA polymerase of the invention may be used in well known DNA sequencing (dideoxy DNA sequencing, cycle DNA sequencing of plasmid DNAs, etc.) and DNA amplification reactions.
REFERENCES:
patent: 4795699 (1989-01-01), Tabor et al.
patent: 4889818 (1989-12-01), Gelfand et al.
patent: 5047342 (1991-09-01), Chatterjee et al.
patent: H1531 (1996-05-01), Blumenthals et al.
patent: 5614365 (1997-03-01), Tabor et al.
patent: 5624833 (1997-04-01), Gelfand et al.
patent: 5912155 (1999-06-01), Chatterjee et al.
patent: 5939301 (1999-08-01), Hughes, Jr. et al.
patent: 5948614 (1999-09-01), Chatterjee
patent: 6001645 (1999-12-01), Slater et al.
patent: 6015668 (2000-01-01), Hughes et al.
patent: 6017745 (2000-01-01), Minkley, Jr.
patent: 6077664 (2000-06-01), Slater et al.
patent: 6306588 (2001-10-01), Solus et al.
patent: 0 655 506 (1995-05-01), None
patent: WO 92/03556 (1992-03-01), None
patent: WO 92/06200 (1992-04-01), None
patent: WO 92/06202 (1992-04-01), None
patent: WO 96/38568 (1996-12-01), None
patent: WO 96/41014 (1996-12-01), None
Bergquist, P.L. et al., “Genetics and Potential Biotechnological Applications of Thermophilic and Extremely Thermophilic Microorganisms,”Biotech. Genet. Eng. Rev. 5:199-244 (1987).
Bernad, A. et al., “A Conserved 3′-5′ Exonuclease Active Site in Prokaryotic and Eukaryotic DNA Polymerases,”Cell 59:219-228 (1989).
Braithwaite, D.K. et al., “Compilation, alignment, and phylogenetic relationships of DNA polymerases,”Nuc. Acids Res. 21:787-802 (1993).
Chien, A. et al., “Deoxyribonucleic Acid Polymerase from Extreme ThermophileThermus aquaticus,” J. Bacteriol. 127:1550-1557 (1976).
Darzins, A. and Chakrabarty, A.M. “Cloning of Genes Controlling Alginate Biosynthesis from a Mucoid Cystic Fibrosis Isolate ofPseudomonas aeruginosa,” J. Bacteriol. 159:9-18 (1996).
Derbyshire, V. et al., “The 3′-5′ exonuclease of DNA polymerase I ofEscherichia coli:contribution of each amino-acid at the active site to the reaction”,EMBO J. 10:17-24 (1990).
Derbyshire, V. et al., “Genetic and Crystallographic Studies of the 3′,5′-Exonucleolytic Site of DNA Polymerase I,”Science 240:199-201 (1988).
Elie, C. et al., “Thermostable DNA polymerase from the archaebacteriumSulfolobus acidocaldarius:Purification, characterization, and immulogical properties,”Eur. J. Biochem. 178:619-626 (1989).
Gutman, D. et al., “Conserved sites in the 5′-3′ exonuclease domain ofEscherichia coli
Chatterjee Deb K.
Hughes, Jr. A. John
Hutson Richard
Invitrogen Corporation
Prouty Rebecca E.
Sterne Kessler Goldstein & Fox P.L.L.C.
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