Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-04-16
2003-06-17
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S007320, C435S071100, C435S091200, C530S350000
Reexamination Certificate
active
06579703
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to a heat shock protein from
Pyrococcus furiosus,
to a method of protecting and extending the durability of a recombinant DNA polymerase, and to a PCR kit.
2. Description of the Related Art
All organisms respond to elevated temperature by specifically inducing the expression of a set of new proteins, termed “heat shock proteins” or “HSPs.” Although this response has been known for over thirty years, the specific role of individual HSPs in the overall response is still largely unknown. The HSPs to which functional character has been attributed have been characterized as molecular chaperones that enable protein folding, preventing denaturation of other proteins, or mediating proteolysis. This role, however, has only been demonstrated for a few of the many known HSPs and the function of other HSPs remains unknown. Moreover, it is not known which of the HSPs are essentials for the overall shock response except in the cases described below.
All organisms have a basal level of thermotolerance that is an organism-specific temperature threshold above which the organisms die. Basal levels of thermotolerance are probably determined by a variety of factors, including, for example, membrane composition and the innate thermal stability of enzymes involved in normal cellular processes. An additional level of thermotolerance can be acquired by exposure of an organism to sublethal processes. Such “acquired thermotolerance” is believed to result from the production of HSPs in response to the sublethal high temperature exposure.
HSPs have been categorized by size and DNA sequence into families that are evolutionarily conserved. These families include the HSP 100, HSP 90, HSP 70, the HSP 60 and a variable class of low molecular weight proteins that range from 12-42 kDa. The HSPs found in this class of low molecular weight proteins are referred to as small heat shock proteins or sHSPs. In animals, this class of low molecular weight proteins ranges from 20-25 kDA. In the plant kingdom, the corresponding range is 14-20 kDa.
All of the low molecular weight HSPs are distinguished by conserved carboxy termini that are highly homologous to the &agr;B-crystallin structural protein of the eye lens. &agr;B-crystallin is itself capable of acting as a molecular chaperone, and all sHSPs have been demonstrated to exhibit chaperone activities in in vitro experiments. Their role in cells has not yet been demonstrated.
While it might be assumed that the HSPs play a role in thermotolerance because of the correlation of their abundant synthesis with exposure to increased temperature, earlier work with yeast had suggested that they are unimportant for the development of thermotolerance, as elimination of a single yeast sHSP had no effect on thermotolerance. In addition, in Drosophilia cells, the use of antisense technology caused a specific decrease in the synthesis of the sHSP 26 protein, but such decrease had no effect on thermotolerance.
In addition to being induced by temperature stress, many HSPs, including those in the sHSP class, can be induced by other stresses such as exposure to arsenite, ethanol, heavy metals, amino acid analogs (Lee, Y. R., et al., Plant Physio. 110:241-48 (1996); and Nover, L., (ed.) Heat Shock Response, CRC Press (1990)) and water stress (Almoguera, C., et al., The Plant Journal 4(6): 947-58 (1993). In addition, increasing numbers of HSPs and HSP homologs are found to be regulated in developmental and tissue-specific ways (see, e.g., Almoguera, C. and J. Jordano, Plant Molecular Biol. 19:781-92 (1992); Apuya, N. R. and J. L. Zimmerman, The Plant Cell, 4:657-65 (1992); Cordewener, J. H. G., et al., Plant Cell 1:1137-1140 (1989). Proteins with highly conserved sequences related to HSPs, HSP cognates, may be expressed in non-stressed normal cells, but are not induced by thermal stress.
The mechanisms of action for the small HSPs are not clearly understood at present. There is a need for a better understanding of sHSPs despite other recombinant archael sHSPs that have been overexpressed in
E. coli.
The present invention embodies an advance in the field of sHSPs that correlatively advances the understanding of the mechanism of sHSPs.
SUMMARY OF THE INVENTION
The invention relates to heat shock proteins and their methods of use.
In one aspect, the invention relates to a purified and isolated nucleic acid sequence encoding a heat shock protein comprising SEQ ID NO. 1.
Another aspect of the invention relates to the protein encoded by the nucleic acid comprising SEQ ID NO. 1, and to compositions comprising same.
Another aspect of the invention relates to a protein comprising the amino acid sequence of SEQ ID NO. 2, and to compositions comprising same.
A still further aspect of the invention relates to a method of protecting and extending the durability of a recombinant DNA polymerase, comprising the steps of:
purifying a low molecular weight heat shock protein;
adding the heat shock protein to a buffer solution containing the polymerase;
incubating the solution at extended temperature for extended time;
adding components necessary for PCR;
thermocycling the reaction to produce product from amplification of genomic deoxyribonucleic acid; and
examining the product of the reaction by gel electrophoresis.
Yet another aspect of the invention relates to a method of maintaining proteins in solution, comprising the steps of:
adding a low molecular weight heat shock protein to the solution;
elevating the temperature of the solution; and
measuring the enzymatic activity by absorbance.
A still further aspect of the invention relates to a PCR kit comprising the protein encoded by the nucleic acid comprising SEQ ID NO. 1, or the protein comprising the amino acid sequence of SEQ ID NO. 2, and one or more other PCR reagents.
In a further compositional aspect, the invention relates to a composition comprising (i) a biological component and (ii) an HSP or a precursor thereof, which is (A) exogenous to the biological component, and (B) thermostabilizingly effective for the biological component in the composition.
The invention contemplates in various further aspects:
a method of enhancing the stability of Taq polymerase in a PCR operation, by conducting the PCR operation in the presence of a HSP;
a PCR kit including PCR primers, Taq polymerase, deoxyribonucleoside triphosphates and an HSP;
transformed cells capable of expressing Pfu-sHSP;
recombinant DNA vectors for expression of Pfu-sHSP; and
a method of stabilizing a protein solution, including a first protein therein, against heat-mediated agglomeration of the first protein in the solution, by incorporating in the solution a heat shock protein that is non-endogenous with respect to the first protein.
Other aspects, features and embodiments of the invention will be more fully apparent from the ensuing disclosure and appended claims.
REFERENCES:
patent: 5474892 (1995-12-01), Jakob et al.
Lee, et al., “Induction and regulation of heat-shock gene expression by an amino acid analog in soybean seedlings,” Plant Physiol. (1996) 110: 241-246.
Jordano, “Tissue-specific expression of sunflower head shock proteins in response to water stress,” The Plant Journal (1993) 4(6), 947-958.
Jordano, “Developmental and environmental concurrent expression of sunflower dry-seed-stored low-molecular-weight heat-shock protein and Lea mRNAs,” Plant Molecular Biology, 19: 781-792, 1992.
Apuya et al., “Heat shock gene expression is controlled primarily at the translational level in carrot cells and somatic embryos,” The Plant Cell, vol. 4, 657-665, Jun. 1992.
Zimmerman, et al., “Novel regulation of heat shock genes during carrot somatic embryo development,” The Plant Cell, vol. 1, 1137-1146, Dec. 1989.
“Heat Shock Response,” ed. Lutz Nover, Ph.D., CRC Press, 1991.
Laksanalamai Pongpan
Robb Frank T.
Fuierer Marianne
Horlick Kenneth R.
Hultquist Steven J.
Spiegler Alexander H.
University of Maryland Biotechnology Institute
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