Thermophilic DNA polymerases from Thermoactinomyces vulgaris

Details Thermophilic DNA polymerases from Thermoactinomyces vulgaris Thermophilic DNA polymerases from Thermoactinomyces vulgaris

2000-03-02

2003-10-14

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S006120, C435S320100, C536S023200

Reexamination Certificate

active

06632645

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to thermostable DNA polymerases derived from the thermophilic eubacterial species
Thermoactinomyces vulgaris
, as well as means for isolating and producing these enzymes. The thermostable DNA polymerases of the present invention are useful in many recombinant DNA techniques, including thermal cycle sequencing, nucleic acid amplification and reverse transcription.
BACKGROUND
Thermophilic bacteria are organisms which are capable of growth at elevated temperatures. Unlike the mesophiles, which grow best at temperatures in the range of 25-40° C., or psychrophiles, which grow best at temperatures in the range of 15-20° C., thermophiles grow best at temperatures greater than 50° C. Indeed, some thermophiles grow best at 65-75° C., and some of the hyperthermophiles grow at temperatures up to 130° C. (e.g., J. G. Black,
Microbiology Principles and Applications
, 2d edition, Prentice Hall, N.J., 1993, p. 145-146).
The thermophilic bacteria encompass a wide variety of genera and species. There are thermophilic representatives included within the phototrophic bacteria (i.e., the purple bacteria, green bacteria, and cyanobacteria), eubacteria (i.e., Bacillus, Clostridium, Thiobacillus, Desulfotomaculum, Thermus, lactic acid bacteria, actinomycetes, spirochetes, and numerous other genera), and the archaebacteria (i.e., Pyrococcus, Thermococcus, Thermoplasma, Thermotoga, Sulfolobus, and the methanogens). There are aerobic, as well as anaerobic thermophilic organisms. Thus, the environments in which thermophiles may be isolated vary greatly, although all of these organisms are isolated from areas associated with high temperatures. Natural geothermal habitats have a worldwide distribution and are primarily associated with tectonically active zones where major movements of the earth's crust occur. Thermophilic bacteria have been isolated from all of the various geothermal habitats, including boiling springs with neutral pH ranges, sulfur-rich acidic springs, and deep-sea vents. In general, the organisms are optimally adapted to the temperatures at which they are living in these geothemal habitats (T. D. Brock, “Introduction: An overview of the thermophiles,” in T. D. Brock (ed.),
Thermophiles: General, Molecular and Applied Microbiology
, John Wiley & Sons, New York, 1986, pp. 1-16). Basic, as well as applied research on thermophiles has provided some insight into the physiology of these organisms, as well as use of these organisms in industry and biotechnology.
I. Uses For Thermophilic Enzymes
Advances in molecular biology and industrial processes have led to increased interest in thermophilic organisms. Of particular interest has been the development of thermophilic enzymes for use in industries such as the detergent, flavor-enhancing, and starch industries. Indeed, the cost savings associated with longer storage stability and higher activity at higher temperatures of thermophilic enzymes, as compared to mesophilic enzymes, provide good reason to select and develop thermophilic enzymes for industrial and biotechnology applications. Thus, there has been much research conducted to characterize enzymes from thermophilic organisms. However, some thermophilic enzymes have less activity than their mesophilic counterparts under similar conditions at the elevated temperatures used in industry (typically temperatures in the range of 50-100° C.) (T. K. Ng and William R. Kenealy, “Industrial Applications of Thermostable Enzymes,” in T. D. Brock (ed.),
Thermophiles: General, Molecular, and Applied Microbiology
, 1986, John Wiley & Sons, New York, pp. 197-215). Thus, the choice of a thermostable enzyme over a mesophilic one may not be as beneficial as originally assumed. However, much research remains to be done to characterize and compare thermophilic enzymes of importance (e.g., polymerases, ligases, kinases, topoisomerases, restriction endonucleases, etc.) in areas such as molecular biology .
II. Thermophilic DNA Polymerases
Extensive research has been conducted on isolation of DNA polymerases from mesophilic organisms such as
E. coli
. (e.g., Bessman et al., J. Biol. Chem. 223:171,1957; Buttin and Kornberg, J. Biol. Chem. 241:5419, 1966; and Joyce and Steitz, Trends Biochem. Sci., 12:288-292, 1987). Other mesophilic polymerases have also been studied, such as those of
Bacillus licheniformis
(Stenesh and McGowan, Biochim. Biophys. Acta 475:32-44, 1977; Stenesh and Roe, Biochim. Biophys. Acta 272:156-166, 1972);
Bacillus subtilis
(Low et al., J. Biol. Chem., 251:1311, 1976; and Ott et al., J. Bacteriol., 165:951, 1986);
Salmonella typhimurium
(Harwood et al., J. Biol. Chem., 245:5614, 1970; Hamilton and Grossman, Biochem., 13:1885, 1974);
Streptococcus pneumoniae
(Lopez et al., J. Biol. Chem., 264:4255, 1989); and
Micrococcus luteus
(Engler and Bessman, Cold Spring Harbor Symp., 43:929, 1979), to name but a few.
Somewhat less investigation has been performed on the isolation and purification of DNA polymerases from thermophilic organisms. However, native (i.e., non-recombinant) and/or recombinant thermostable DNA polymerases have been purified from various organisms, as shown in Table 1 below.
TABLE 1
Polymerase Isolation From Thermophilic Organisms
Organism
Citation
Thermus aquaticus
Kaledin et al., Biochem., 45:494-501 (1980);
Biokhimiya 45:644-651 (1980).
Chien et al., J. Bacteriol., 127:1550 (1976).
University of Cincinnati Master's thesis by A.
Chien, “Purification and Characterization of
DNA Polymerase from
Thermus aquaticus,
”
(1976).
University of Cincinnati, Master's thesis
by D. B. Edgar, “DNA Polymerase From an
Extreme Thermophile:
Thermus aquaticus,
”
(1974).
U.S. Pat. No. 4,889,818*
U.S. Pat. No. 5,352,600*
U.S. Pat. No. 5,079,352*
European Patent Pub. No. 258,017*
PCT Pub. No. WO 94/26766*
PCT Pub. No. WO 92/06188*
PCT Pub. No. WO 89/06691*
Thermatoga
PCT Pub. No. WO 92/03556*
maritima
Thermatoga
U.S. Pat. No. 5,912,155*
neapolitana
U.S. Pat. No. 5,939,301*
U.S. Pat. No. 6,001,645*
Thermotoga strain
Simpson et al., Biochem. Cell Biol.,
FjSS3-B.1
68:1292-1296 (1990).
Thermosipho
PCT Pub. No. 92/06200*
africanus
U.S. Pat. No. 5,968,799*
Thermus
Myers and Gelfand, Biochem., 30:7661 (1991).
thermophilus
PCT Pub. No. WO 91/09950*
PCT Pub. No. WO 91/09944*
Bechtereva et al., Nucleic Acids Res.,
17:10507 (1989).
Glukhov et al., Mol. Cell. Probes
4:435-443 (1990).
Carballeira et al., BioTech.,
9:276-281 (1990).
Rüttiman et al., Eur. J.
Biochem., 149:41-46 (1985).
Oshima et al., J. Biochem., 75:179-183 (1974).
Sakaguchi and Yajima, Fed. Proc., 33:1492
(1974) (abstract).
Thermus flavus
Kaledin et al., Biochem., 46:1247-1254 (1981);
Biokhimiya 46:1576-1584 (1981).
PCT Pub No. WO 94/26766*
Thermus ruber
Kaledin et al., Biochem., 47:1515-1521 (1982);
Biokhimiya 47:1785-1791 (1982).
Thermoplasma
Hamal et al., Eur. J. Biochem., 190:517-521 (1990).
acidophilum
Forterre et al., Can. J. Microbiol.,
35:228-233 (1989).
Sulfolobus
Salhi el al., J. Mol. Biol., 209:635-641 (1989).
acidocaldarius
Salhi et al., Biochem. Biophys. Res. Comm.,
167:1341-1347 (1990).
Rella et al., Ital. J. Biochem., 39:83-99 (1990).
Forterre et al., Can. J. Microbiol.,
35:228-233 (1989).
Rossi et al., System. Appl. Microbiol.,
7:337-341 (1986).
Klimczak et al., Nucleic Acids Res.,
13:5269-5282 (1985).
Elie et al., Biochim. Biophys. Acta
951:261-267 (1988).
Bacillus caldotenax
J. Biochem., 113:401-410 (1993).
Bacillus
Sellmann et al., J. Bacteriol.,
stearothermophilus
174:4350-4355 (1992).
Stenesh and McGowan, Biochim. Biophys. Acta
475:32-44 (1977).
Stenesh and Roe, Biochim. Biophys. Acta
272:156-166 (1972).
Kaboev et al., J. Bacteriol.,
145:21-26 (1981).
Methanobacterium
Klimczak et al., Biochem.,
thermoautotropicum
25:4850-4855 (1986).
Thermococcus
Kong et al., J. Biol. Chem. 268:1965 (1993)
litoralis
U.S. Pat. No. 5,210,036*
U.S. Pat. No. 5,322,785*
Anaerocellum
Ankenbauer et al., WO 98/14588*
thermophilus
Pyrococcus sp.
U.S. Pat. No. 6,008,025*
KOD
1
Pyrococcus furiosus
Lundberg et al., Gene 108:1 (1991)
PCT Pub. WO 92/09689
U.S. Pat. No. 5,948,

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