Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-01-14
2001-06-12
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C435S183000, C435S194000, C530S350000
Reexamination Certificate
active
06245533
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the fields of molecular biology and protein chemistry. Specifically, the invention provides compositions comprising thermostable enzymes, particularly thermostable DNA polymerases and restriction endonucleases, that are substantially free from contamination by nucleic acids, and methods for the production of such thermostable enzymes. Compositions comprising the thermostable enzymes of the present invention may be used in a variety of applications, including amplification and sequencing of nucleic acids.
BACKGROUND OF THE INVENTION
A variety of techniques have been traditionally employed to facilitate the preparation of intracellular proteins from microorganisms. Typically, the initial steps in these techniques involve lysis, rupture or permeabilization of the bacterial cells, to disrupt the bacterial cell wall and allow release of the intracellular proteins into the extracellular milieu. Following this release, the desired proteins are purified from the extracts, typically by a series of chromatographic steps.
Several approaches have proven useful in accomplishing the release of intracellular proteins from bacterial cells. Included among these are the use of chemical lysis or permeabilization, physical methods of disruption, or a combination of chemical and physical approaches (reviewed in Felix, H.,
Anal. Biochem
. 120:211-234 (1982)).
Chemical methods of disruption of the bacterial cell wall that have proven useful include treatment of cells with organic solvents such as toluene (Putnam, S. L., and Koch, A. L.,
Anal. Biochem
. 63:350-360 (1975); Laurent, S. J., and Vannier, F. S.,
Biochimie
59:747-750 (1977); Felix, H.,
Anal. Biochem
. 120:211-234 (1982)), with chaeotropes such as guanidine salts (Hettwer, D., and Wang, H.,
Biotechnol. Bioeng
. 33:886-895 (1989)), with antibiotics such as polymyxin B (Schupp, J. M., et al.,
BioTechniques
19:18-20 (1995); Felix, H.,
Anal. Biochem
. 120:211-234 (1982)), or with enzymes such as lysozyme or lysostaphin (McHenry, C. S, and Kornberg, A,
J. Biol. Chem
. 252(18):6478-6484 (1977); Cull, M., and McHenry, C. S.,
Meth. Enzymol
. 182:147-153 (1990); Hughes, A. J., Jr., et al.,
J. Cell. Biochem. Suppl
. 0 16(Part B):84 (1992); Sambrook, J., et al., in:
Molecular Cloning: A Laboratory Manual
, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, p. 17.38 (1989); Ausubel, F. M., et al., in:
Current Protocols in Molecular Biology
, New York: John Wiley & Sons, pp. 4.4.1-4.4.7 (1993)). The permeabilization effects of these various chemical agents may be enhanced by concurrently treating the bacterial cells with detergents such as TRITON X-100®, sodium dodecylsulfate (SDS) or Brij 35 (Laurent, S. J., and Vannier, F.S.,
Biochimie
59:747-750 (1977); Felix, H.,
Anal. Biochem
. 120:211-234 (1982); Hettwer, D., and Wang, H.,
Biotechnol. Bioeng
. 33:886-895 (1989); Cull, M., and McHenry, C. S.,
Meth. Enzymol
. 182:147-153 (1990); Schupp, J. M., et al.,
BioTechniques
19:18-20 (1995)), or with proteins or protamines such as bovine serum albumin or spermidine (McHenry, C. H., and Kornberg, A,
J. Biol. Chem
. 252(18):6478-6484 (1977); Felix, H.,
Anal. Biochem
. 120:211-234 (1982); Hughes, A. J., Jr., et al.,
J. Cell. Biochem. Suppl
. 0 16(Part B):84 (1992)).
In addition to these various chemical treatments, a number of physical methods of disruption have been used. These physical methods include osmotic shock, e.g., suspension of the cells in a hypotonic solution in the presence or absence of emulsifiers (Roberts, J. D., and Lieberman, M. W.,
Biochemistry
18:4499-4505 (1979); Felix, H.,
Anal. Biochem
. 120:211-234 (1982)), drying (Mowshowitz, D. B.,
Anal. Biochem
. 70:94-99 (1976)), bead agitation such as ball milling (Felix, H.,
Anal. Biochem
. 120:211-234 (1982); Cull, M., and McHenry, C. S.,
Meth. Enzymol
. 182:147-153 (1990)), temperature shock, e.g., freeze-thaw cycling (Lazzarini, R. A., and Johnson, L. D.,
Nature New Biol
. 243:17-20 (1975); Felix, H.,
Anal. Biochem
. 120:211-234 (1982)), sonication (Amos, H., et al.,
J. Bacteriol
. 94:232-240 (1967); Ausubel, F. M., et al., in:
Current Protocols in Molecular Biology
, New York: John Wiley & Sons, pp. 4.4.1-4.4.7 (1993)) and pressure disruption, e.g, use of a french pressure cell (Ausubel, F. M., et al., in:
Current Protocols in Molecular Biology
, New York: John Wiley & Sons, pp. 16.8.6-16.8.8 (1993)). Other approaches combine these chemical and physical methods of disruption, such as lysozyme treatment followed by sonication or pressure treatment, to maximize cell disruption and protein release (Ausubel, F. M., et al., in:
Current Protocols in Molecular Biology
, New York: John Wiley & Sons, pp. 4.4.1-4.4.7 (1993)).
These disruption approaches have several advantages, including their ability to rapidly and completely (in the case of physical methods) disrupt the bacterial cell such that the release of intracellular proteins is maximized. In fact, these approaches have been used in the initial steps of processes for the purification of a variety of bacterial cytosolic enzymes, including natural and recombinant proteins from mesophilic organisms such as
Escherichia coli, Bacillus subtilis
and
Staphylococcus aureus
(Laurent, S. J., and Vannier, F. S.,
Biochimie
59:747-750 (1977); Cull, M., and McHenry, C. S.,
Meth. Enzymol
. 182:147-153 (1990); Hughes, A. J., Jr., et al.,
J. Cell. Biochem. Suppl
. 0 16(Part B):84 (1992); Ausubel, F. M., et al., in:
Current Protocols in Molecular Biology
, New York: John Wiley & Sons, pp. 4.4.1-4.4.7 (1993)), as well as phosphatases, restriction enzymes, DNA or RNA polymerases and other proteins from thermophilic bacteria and archaea such as
Thermus aquaticus, Thermus thermophilus, Thermus flavis, Thermus caldophilus, Thermotoga maritima, and Sulfolobus acidocaldarius
(Shinomiya, T., et al.,
J. Biochem
. 92(6):1823-1832 (1982); Elie, C., et al.,
Biochim. Biophys. Acta
951(2-3):261-267 (1988): Palm, P., et al.,
Nucl. Acids Res
. 21(21):4904-4908 (1993); Park, J. H., et al.,
Eur. J. Biochem
. 214(1):135-140 (1993); Harrell, R. A., and Hand, R.P.,
PCR Meth. Appl
. 3(6):372-375 (1994); Meyer, W., et al.,
Arch. Biochem. Biophys
. 319(1):149-156 (1995)).
However, these methods possess distinct disadvantages as well. For example, the physical methods by definition involve shearing and fracturing of the bacterial cell walls and plasma membranes. These processes thus result in extracts containing large amounts of particulate matter, such as membrane or cell wall fragments, which must be removed from the extracts, typically by centrifugation, prior to purification of the enzymes. This need for centrifugation limits the batch size capable of being processed in a single preparation to that of available centrifuge space; thus, large production-scale preparations are impracticable if not impossible. Furthermore, physical methods, and many chemical permeation techniques, typically result in the release from the cells not only of the desired intracellular proteins, but also of undesired nucleic acids and membrane lipids (the latter particularly resulting when organic solvents are used to permeabilize the cells). These undesirable cellular components also complicate the subsequent processes for purification of the desired proteins, as they increase the viscosity of the extracts (Sambrook, J., et al., in:
Molecular Cloning: A Laboratory Manual
, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, p. 17.38 (1989); Cull, M., and McHenry, C. S.,
Meth. Enzymol
. 182:147-153 (1990)), and bind with high avidity and affinity to nucleic acid-binding proteins such as DNA polymerases, RNA polymerases and restriction enzymes.
These limitations have been partially overcome in the case of proteins prepared from mesophilic bacteria. For example, mild chemical disruption of
E. coli, B. subtilis
and
Salmonella typhimurium
has been conducted to permeabilize these cells, allowing free mobility of proteins across the membrane of the cells or resultant
Goldstein Adam S.
Hughes, Jr. A. John
Horlick Kenneth R.
Invitrogen Corporation
Sterne Kessler Goldstein & Fox P.L.L.C.
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