Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-12-23
2001-01-16
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S071100, C530S412000
Reexamination Certificate
active
06174704
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an improved method for the recovery of proteins prepared by recombinant DNA procedures. More particularly, the invention relates to an improved method for the separation and recovery of such proteins from the cellular host in which they have been expressed.
BACKGROUND OF THE INVENTION
Recombinant DNA technology makes it possible to produce large amounts of desired proteins in host cells. To recover a recombinantly produced protein expressed in a host cell, it is first necessary to lyse the cell and then extract the protein from the cellular components. The lysis method should not be harmful to the native characteristics of the protein of interest.
Sonication, French press and enzymatic lysis, either alone or in combination with detergents, are currently available techniques used for cell lysis and protein isolation from host cells. These techniques, however, either require special instrumentation that may not always be available or have other limitations, such as being time consuming or generating heat which can denature certain proteins. Also, the methods, in general, do not permit the recovery of soluble recombinant protein in high yields.
In addition, the available methods do not allow for the optimum recovery of the protein of interest from precipitated dense, granular protein forms, known as inclusion bodies, that are distributed throughout the cytoplasm of the cell. In the expression of some proteins, the inclusion bodies contain high levels of the protein of interest and they can be easily separated from other cytoplasmic proteins by centrifugation. However, using current techniques, it is difficult to extract and purify the protein of interest from the inclusion bodies.
SUMMARY OF THE INVENTION
Now, in accordance with the present invention, there is provided an improved method for the preparation and extraction of a protein of interest, prepared by recombinant DNA techniques, from the host cell in which it is expressed. The improvement finds application in the general method of preparing and isolating a protein from its host cell which involves the steps of expressing the protein in the host cell, lysing the cell to release the protein from the cell, and extracting the protein of interest from other host cell components. The improvement provided by the present invention resides in the use of an aqueous reagent solution consisting essentially of an alkylglycoside or alkythioglycoside to lyse the cell and concurrent therewith to extract the protein of interest from other host cellular components.
The improved method described herein is non-mechanical and accomplished under physiological conditions. Accordingly, the method is gentle and non-disruptive to the protein of interest. It is rapid and easy to accomplish since lysing and extraction occur with the use of a single reagent solution. Of particular significance is the fact that, by practicing the present invention, the protein of interest can be recovered in a higher yield with less manipulative steps than with conventional methods. A related advantage accompanying use of the present invention is that inclusion bodies can be recovered from cellular cytoplasm in a purified form.
REFERENCES:
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Rodeghiero et al. Platelet Von Willebrand Factor Assay: Results using Two Methods for Platelet Lysis. Thromb. Res. 59: 259-267, 1990.
Saito et al. Characteristics of n-octyl D-thioGlucopyroanoside, a new Non-ionic Detergent useful for Membrane Biochemistry. 222: 829-832, 1984.
Rosenberg, I.M. Protein Analysis and Purification: Benchtop Techniques. Birkhauser, Boston. p. 108-109, 1996.
Burden et al. Biotechnology: Proteins to PCR. Birkhauser, Boston. p. 69-71, 1995.
Schutte et al. Pilot-and Process-Scale Techniques for Cell Disruption. Biotechnol. Appl. Biochem. 12: 599-620, 1990.
Chu Ruiyin
Mallia A. Krishna
Low Christopher S. F.
Pierce Chemical Company
Schnizer Holly
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