Purified thermostable Pyrococcus furiosus DNA polymerase I

Details Purified thermostable Pyrococcus furiosus DNA polymerase I Purified thermostable Pyrococcus furiosus DNA polymerase I

1999-02-05

2002-12-03

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S471000, C435S006120, C435S325000, C435S320100, C435S252300, C536S023100, C536S023200

Reexamination Certificate

active

06489150

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a thermostable enzyme having DNA polymerase I activity useful in nucleic acid synthesis by primer extension reaction.
BACKGROUND
The archaebacteria are a recently discovered group of microorganisms that grow optimally at temperatures above 80° C. Some 20 species of these extremely thermophilic bacteria-like organisms have been isolated, mainly from shallow submarine and deep sea geothermal environments. Most of the archaebacteria are strict anaerobes and depend on the reduction of elemental sulfur for growth.
The archaebacteria include a group of “hyperthermophiles” that grow optimally around 100° C. These are presently represented by three distinct genera, Pyrodictium, Pyrococcus, and Pyrobaculum.
Phyodictium brockii
(T
opt
105° C.) is an obligate autotroph which obtains energy be reducing S
0
to H
2
S with H
2
, while
Pyrobaculum islandicum
(T
opt
100° C.) is a faculative heterotroph that uses either organic substrates or H
2
to reduce S
0
. In contrast,
Pyrococcus furiosus
(T
opt
100° C) grows by a fermentative-type metabolism rather than by S
0
respiration. It is a strict heterotroph that utilizes both simple and complex carbohydrates where only H
2
and CO
2
are the detectable products. The organism reduces elemental sulfur to H
2
S apparently as a form of detoxification since H
2
inhibits growth.
The discovery of microorganisms growing optimally around 100° C. has generated considerable interest in both academic and industrial communities. Both the organisms and their enzymes have the potential to bridge the gap between biochemical catalysis and many industrial chemical conversions. However, knowledge of the metabolism of the hyperthermophilic microorganisms is presently very limited.
The polymerase chain reaction (PCR) is a powerful method for the rapid and exponential amplification of target nucleic acid sequences. PCR has facilitated the development of gene characterization and molecular cloning technologies including the direct sequencing of PCR amplified DNA, the determination of allelic variation, and the detection of infectious and genetic disease disorders. PCR is performed by repeated cycles of heat denaturation of a DNA template containing the target sequence, annealing of opposing primers to the complementary DNA strands, and extension of the annealed primers with a DNA polymerase. Multiple PCR cycles result in the exponential amplification of the nucleotide sequence delineated by the flanking amplification primers.
An important modification of the original PCR technique was the substitution of
Thermus aguaticus
(Taq) DNA polymerase in place of the Klenow fragment of
E. coli
DNA pol I (Saiki, et al.
Science
, 230:1350-1354 (1988)). The incorporation of a thermostable DNA polymerase into the PCR protocol obviates the need for repeated enzyme additions and permits elevated annealing and primer extension temperatures which enhance the specificity of primer:template associations. Taq polymerase thus serves to increase the specificity and simplicity of PCR.
Although Taq polymerase is used in the vast majority of PCR performed today, it has a fundamental drawback: purified Taq DNA polymerase enzyme is devoid of 3′ to 5′ exonuclease activity and thus cannot excise misinserted nucleotides (Tindall, et al.,
Biochemistry
, 29:5226-5231 (1990)). Several independent studies suggest that 3′ to 5′ exonuclease-dependent proofreading enhances the fidelity of DNA synthesis. Reyland et al,
J. Biol. Chem
., 263:6518-6524, 1988; Kunkel et al,
J. Biol. Chem
., 261:13610-13616, 1986; Bernad et al,
Cell
, 58:219-228, 1989. Consistent with these findings, the observed error rate (mutations per nucleotide per cycle) of Taq polymerase is relatively high; estimates range from 2×10
−4
during PCR (Saiki et al.,
Science
, 239:487-491 (1988); Keohavaong et al.
Proc. Natl. Acad. Sci. USA
, 86:9253-9257 (1989)) to 2×10
−5
for base substitution errors produced during a single round of DNA synthesis of the lacZ gene (Eckert et al.,
Nucl. Acids Res
., 18:3739-3744 (1990)).
Polymerase induced mutations incurred during PCR increase arithmetically as a function of cycle number. For example, if an average of two mutations occur during one cycle of amplification, 20 mutations will occur after 10 cycles and 40 will occur after 20 cycles. Each mutant and wild type template DNA molecule will be amplified exponentially during PCR and thus a large percentage of the resulting amplification products will contain mutations. Mutations introduced by Taq polymerase during DNA amplification have hindered PCR applications which require high fidelity DNA synthesis.
SUMMARY OF THE INVENTION
A thermostable DNA polymerase from the hyperthermophilic, marine archaebacterium, Pyrococcus furiosus (Pfu) has been discovered. The monomeric, multifunctional enzyme possesses both DNA polymerase and 3′ to 5′ exonuclease activities. The polymerase is extremely thermostable with a temperature optimum near 75° C. The purified enzyme functions effectively in the polymerase chain reaction (PCR). In addition, results from PCR fidelity studies indicate that Pyrococcus furiosus DNA polymerase yields amplification products containing 12 fold less mutations than reaction products from similar amplifications performed with Taq DNA polymerase. The 3′ to 5′ exonuclease dependent proofreading activity of Pfu DNA polymerase will excise mismatched 3′ terminal nucleotides from primer:template complexes and correctly incorporate nucleotides complementary to the template strand.
Unlike Taq DNA polymerase, Pfu DNA polymerase does not possess 5′ to 3′ exonuclease activity. Pfu, like Taq and Vent polymerases, does exhibit a polymerase dependent 5′ to 3′ strand displacement activity. Pfu DNA polymerase remains greater that 95% active after one hour incubation at 95° C. In contrast, Vent polymerase [New England Biolabs (NEB) Beverly, Mass.] looses greater than 50% of its polymerase activity after one hour incubation at 95° C. Pfu DNA polymerase is thus unexpectedly superior to Taq and Vent DNA polymerases in amplification protocols requiring high fidelity DNA synthesis.
Thus, the present invention contemplates a purified thermostable
P. furiosus
DNA polymerase I (Pfu DNA Pol I or Pyro polymerase) having an amino terminal amino acid residue sequence represented by the formula shown in SEQ ID NO 1, having 775 amino acid residues.
The apparent molecular weight of the native protein is about 90,000-93,000 daltons as determined by SDS-PAGE under non-denaturing (non-reducing) conditions using Taq polymerase as a standard having a molecular weight of 94,000 daltons. In preferred embodiments, the Pyro polymerase is isolated from
P. furiosus
, and more preferably has a specific 3′ to 5′ exonuclease activity.


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G.M. Wahl et al. “Molecular Hybridization of Immobilized Nucleic Acids: Theorectical Concepts and Practical Considerations”, Methods in Enzymol. 152: 399-415. (1987).*
Bernad et al., “A Conserved 3′→5′ Exonuclease Active Site in Prokaryotic and Eukaryotic DNA Polymerases,”Cell59:219-228 (1989).
Berger, et al., “Guide to Molecular Cloning Techniques,”Meth. In Enzymol. 152:393-399; 415-423, 432-449, 661-704 (1987).
Blanco, et al., “Evidence favouring the hypothesis of a conserved 3′→5′ exonuclease active site in DNA-dependent DNA polymerases,”Gene112:139-144 (1992).
Bryant et al.,J. Biol. Chem., 264:5070-5079 (1989).
Deutscher, “Guide to Protein Purification,”Meth. In Enzymol. 182:738-751 (1990).
Dutton et al., “General method for amplifying regions of very high G+C content,”Nucl. Acids Res. 2953-2954 (1993).
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