Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
2000-03-30
2002-03-19
Salimi, Ali R. (Department: 1648)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C435S188000, C435S069100, C435S069200, C435S068100, C435S071100, C435S071200
Reexamination Certificate
active
06358718
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides methods for stabilizing proteins, preventing protein aggregation, renaturation of previously-denatured proteins, reactivating a protein that has been inactivated by denaturation, preserving enzyme activities under conditions of elevated temperatures, inducing thermotolerance in bacteria, increasing the temperature optimum for the activity of an enzyme, and preventing formation of inclusion bodies by bacterially-expressed recombinant proteins.
2. Background Art
Technologies for the production of virtually any polypeptide by introduction, by recombinant DNA methods, of a natural or synthetic DNA fragment coding for this particular polypeptide into a suitable host have been under intense development over the past fifteen years, and are at present essential tools for biochemical research and for a number of industrial processes for production of high-grade protein products for biomedical or other industrial use.
The production of recombinant bioactive protein has become possible through developments in the field of recombinant DNA technology, but is often not successful in practice because frequently folding and/or secretion of the heterologous proteins that are to be produced does not occur correctly or occurs inefficiently. Such a problem usually occurs in cases of strong expression of the heterologous protein in a cell which has insufficient capacity to ensure correct folding or secretion of a protein that is produced in a large amount. The problem arises from the fact that recombinant proteins are produced in large amounts in prokaryotic cells or eukaryotic cells so that the activity of the cellular folding proteins is not sufficient for folding the huge amount of foreign protein.
A recombinant gene product which is foreign to the cell or is produced at high levels often activates cellular defense mechanisms similar to those activated by heat shock or exposure to toxic amino acid analogues, pathways that have been designed by nature to help the cell to get rid of “wrong” polypeptide material by controlled intracellular proteolysis or by segregation of unwanted polypeptide material into storage particles (“inclusion bodies”). The recombinant protein in these storage particles is often deposited in a misfolded and aggregated state, in which case it becomes necessary to dissolve the product under denaturing and reducing conditions and then fold the recombinant polypeptide by in vitro methods to obtain a useful protein product. Another problem is that proteins often tend to aggregate in solution. The formation of aggregates then causes loss in the activity of biologically active proteins or prevents the correct folding of unfolded or misfolded proteins by in vitro refolding methods.
In nature, correct folding of proteins produced by a cell is ensured by molecular chaperone proteins which are essential for correct functioning of cells as they ensure that proteins produced by the cells are folded in a correct manner. Specific chaperone proteins have been described for protein transport from the cell cytoplasm to cell organelles such as the mitochondrion, the chloroplast, the cell nucleus and the ER. The production of many of these proteins is induced in cells that are subjected to a heat shock and chaperone proteins are therefore often called “heat-shock proteins”.
The present invention offers a solution to the problems of aggregation and formation of inclusion bodies, and also provides a means for increasing thermal tolerance of a protein (e.g., by stabilizing a thermolabile enzyme) and for preserving enzyme activity under denaturing conditions, in that methods are provided for the use of protein B23 as a chaperone in vitro or in vivo. The use of B23 as chaperone allows efficient production of proteins in a transformed host cell by co-expression of B23 with the desired, or by treating protein with B23 in vitro.
The present invention is based on the surprising discovery that B23 has a chaperone function which can be used in vitro or in vivo for deaggregation and prevention of aggregation of a protein, and in vivo for the efficient production of recombinant protein from a host cell if B23 and the recombinant protein are co-expressed in the cell.
SUMMARY OF THE INVENTION
In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to a method of preventing protein aggregation of a selected protein, comprising contacting a solution containing the selected protein with the nucleolar protein B23, thereby promoting solubility of the selected protein.
In another embodiment, the invention provides a method of protecting a selected protein from aggregation during denaturation, comprising contacting the selected protein with the nucleolar protein B23 while subjecting the selected protein to at least one denaturing condition, thereby preventing aggregation of the selected protein.
The invention also provides a method of preserving enzyme activity of a selected enzyme, comprising contacting the selected enzyme with nucleolar protein B23.
In yet another embodiment, the invention provides a method of renaturing a denatured protein, comprising contacting the denatured protein with nucleolar protein B23 under conditions that promote renaturation of the denatured protein.
The invention also provides a method of inducing thermal tolerance in bacteria, comprising contacting the bacteria with the nucleolar protein B23.
In yet another embodiment, the invention provides a method of preventing the formation of inclusion bodies in recombinant protein-expressing bacteria, comprising promoting expression of the recombinant protein by the bacteria and contacting the newly expressed recombinant protein with nucleolar protein B23.
In yet another embodiment, the invention provides a method of stabilizing a thermolabile enzyme, comprising contacting the thermolabile enzyme with the nucleolar protein B23.
Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
REFERENCES:
Craig E.A, Chaperones, Molecular in Molecular Biology biotechnology edited by Meyers R. A. 1995, pp. 162-165.*
Yon J. M, Protein Aggregation in, Molecular in Molecular Biology biotechnology edited by Meyers R. A. 1995, pp. 728-731.*
Borer et al., “Major Nucleolar Proteins Shuttle between Nucleus and Cytoplasma”Cell56:379-390, Feb. 1989.
Chan et al., “Characterization of the cDNA Encoding Human Nucleophosmin and Studies of Its Role in Normal and Abnormal Growth”Biochemistry28:1033-1039, 1989.
Fankhauser et al., “Specific Complex of Human Immunodeficiency Virus Type 1 Rev and Nucleolar B23 Proteins: Dissociation by the Rev Response Element”Molecular and Cellular Biology11(5):2567-2575, May 1991.
Feuerstein et al., “Identification of a Prominent Nuclear Protein Associated with Proliferation of Normal and Malignant B Cells”Journal of Immunology139(6):1818-1822, Sep. 15, 1987.
Goldfarb, D.S., “Karyophilic Peptides: Applications to the Study of Nuclear Transport”Cell Biology International Reports, 12(9):809-832, Sep. 1988.
Schmidt-Zachmann et al., “A Constitutive Nucleolar Protein Identified as a Member of the Nucleoplasmin Family”The EMBO Journal,6(7):1881-1890, 1987.
Szebeni et al., “Interactions of Nucleolar Protein B23 with Peptides Related to Nuclear Localization Signals”Biochemistry, 34:8037-8042, 1995.
Szebeni et al., “Nucleolar Protein B23 Stimulates Nuclear Import of the HIV-1 Rev Protein and NLS-Conjugated Albumin”Biochemistry, 36(13):3941-3949, 1997.
Szebeni et al., “Molecular Chaperone-Like Activity of
Olson Mark
Szebeni Atilla
Li Bao Qun
Needle & Rosenberg P.C.
Salimi Ali R.
The University of Mississippi Medical Center
LandOfFree
Method for stabilization and renaturation of proteins using... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for stabilization and renaturation of proteins using..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for stabilization and renaturation of proteins using... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2845407