Purified thermostable pyrococcus furiosus DNA ligase

Details Purified thermostable pyrococcus furiosus DNA ligase Purified thermostable pyrococcus furiosus DNA ligase

1997-08-22

2001-08-28

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S183000, C435S320100, C536S023100, C536S023200

Reexamination Certificate

active

06280998

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a thermostable enzyme having DNA ligase activity useful in ligase chain reactions and other nucleic acid manipulations.
BACKGROUND
The hyperthermophiles of archaebacteria are a recently discovered group of microorganisms that grow optimally at temperatures around 100° C. Many 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 “hyperthermophiles” are presently represented by three distinct genera, Pyrodictium, Pyrococcus, and Pyrobaculum.
Pyrodictium brockii
(T
opt
105° C.) is an obligate autotroph which obtains energy by reducing S
0
to H
2
S with H
2
, while
Pyrobaculum islandicum
(T
opt
100° C.) is a facultative 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 ligase chain reaction (LCR) provides a powerful method for the rapid and sensitive amplification of DNA fragments. LCR allows the specific detection of a target nucleic acid sequence with a single base mutation. LCR has facilitated the development of gene diagnostic technologies including the determination of allelic variation, and the detection of infectious and genetic disease disorders.
LCR is performed by repeated cycles of heat denaturation of a DNA template containing the target sequence, annealing a first set of two adjacent oligonucleotide probes to the target DNA sequence in a unique manner, and a second set of complementary oligonucleotide probes that hybridize to the sequence opposite to the target DNA sequence. Thereafter, a thermostable DNA ligase will covalently link each pair of adjacent probes provided there is complete complementarity at the junction of the two adjacent probes. Because the oligonucleotide products from one round may serve as substrates during the next round, the signal is amplified exponentially, analogous to the polymerase chain reaction (PCR).
LCR has been extensively described by Landegren et al.,
Science
, 241:1077-1080 (1988); Wu et al.,
Genomics
, 4:560-569 (1989); Barany, in
PCR Methods and Applications
, 1:5-16 (1991); and Barany,
Proc. Natl. Acad. Sci. USA
, 88:189-193 (1991).
An important aspect of successful LCR is to reduce background target-independent ligations, including blunt-end ligations. Such target-independent ligations produce a product the same size as the desired product from a target-directed LCR reaction, and as such are indistinguishable from the desired reaction product. The method requires a thermostable ligase to allow ligation to occur under temperature conditions that prevent mismatches from hybridizing to form acceptable substrates for a thermostable DNA ligase.
DNA ligases exhibiting limited temperature stability have been isolated from
Thermus aguaticus
(
Taq
), and from
Thermus thermophilus
(
Tth
). See, for example Takahashi et al.,
J. Biol. Chem
., 259:10041-10047 (1984). However, these enzymes do not maintain thermostability at temperatures greater than about 65° C. for prolonged periods of up to 10 to 30 minutes as required for typical LCR protocols. Thus, the known DNA ligases are unstable at high temperatures for prolonged periods, and therefore require a “pre-melt” step in LCR procedures to separate the two strands of the genomic DNA molecule prior to the addition of the enzyme followed by LCR cycles below about 85° C. to 90° C.
There continues to exist a need for a thermostable DNA ligase that can retain activity at high temperatures for prolonged periods of time, such as during ligase chain reactions.
SUMMARY OF THE INVENTION
A thermostable DNA ligase from hyperthermophilic marine archaebacterium species has been discovered. The monomeric enzyme possesses DNA ligase activity and is substantially free from target-independent ligation activity in ligase chain reactions. The ligase is extremely thermostable at 100° Centigrade (C), substantially retaining its catalytic activity after 30 minutes exposure to temperatures of about 85° C. to about 100° C., and has a catalytic activity range of about 30° C. to about 80° C., with an enzymatic activity temperature optimum of about 70° C.
The purified thermostable DNA ligase of this invention functions effectively in the ligase chain reaction (LCR) without catalyzing significant blunt-end ligation, and can be used without the limitations of thermo-instability during exposure to high temperatures during the LCR procedures. A thermostable DNA ligase of this invention can be utilized in LCR without the need to “pre-melt” the genomic DNA prior to LCR.
The apparent molecular weight of the native protein is about 50,000 to 70,000 daltons, and preferably about 55,000 to 65,000 daltons, as determined by SDS-PAGE under denaturing (reducing) conditions. Preferably, the thermostable DNA ligase has DNA ligase activity optimum in a pH range of 6-8, affording a wide range of hybridization conditions in which the enzyme is active. A preferred thermostable DNA ligase also binds rATP.
A preferred thermostable DNA ligase is isolated from
Pyrococcus furiosus
(
Pfu
) and is designated
Pfu
DNA ligase.
The invention also describes a plasmid containing a gene coding for a thermostable DNA ligase which catalyzes template-dependent ligation at temperatures of about 30° C. to about 80° C., and which substantially retains its catalytic activity when subjected to temperatures of from about 85° C. to about 100° C. The plasmid is useful for producing recombinant, purified, thermostable DNA ligase.


REFERENCES:
patent: 5506137 (1996-04-01), Mathur et al.
patent: 373 962 (1990-06-01), None
patent: WO 91/17239 (1991-11-01), None
Barany, F., et. al., “Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene,” Gene 109:1-11 (1991).
Barany, F., “Genetic disease detection and DNA amplification using cloned thermostable DNA ligase,” Proc. Natl. Acad. Sci. USA 88:189-193 (1991).
Barany, F., “The ligase chain reaction on a PCR world,” PCR Methods and Applications, 1:5-16 (1991).
Bond, S., et al. “New methods for detecting HPV,” in Papillomaviruses in Human Pathology (J. Monsonego Ed., 1990), pp. 425-434.
Bryant, F., et al., “Characterization of hydrogenase from the hyperthermophilic Archeabacterium,Pyrococcus furiosus,” J. Biol. Chem. 264:5070-5079 (1989).
Fiala, G., et al., “Pyrococcus furiosussp.nov.represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100°C,” Arch. Microbiol. 145:56-61 (1986).
Hardy, S., et al., “DNA ligase I fromXenopus laeviseggs,” Nucleic Acids Res. 19(4) 701-705 (1991).
Landegren, U., et al., “A Ligase-Medicated Gene Detection Technique,” Science 241:1077-1080 (1988).
Landegren, U., et al., “DNA diagnostics-Molecular Techniques and Automation,” Science 242:229-237 (1988).
Lindahl, T., “DNA ligase from Rabbit Tissues,” Methods in Enzymology 21:333-338 (1971).
Marsh, E., et al., “Pyrococcus furiosusDNA ligase and the ligase chain reaction,” Strategies in Molecular Biology 5:73-76 (1992).
Oishi, N., et al., “Purification and Properties of a DNA Ligase from Sea Urchin Embryos,” J. Biochem 95:1187-1192 (1984).
Panasenko, S.M., et al., “A Simple, Three-Step Procedure for the Large Scale Purification of

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