Method of isolation and purification of fusion polypeptides
AssigneesBoard of Regents, The University of Texas System, State of Oregon, The, Acting By and Through the Oregon State System of Higher Education, on Behalf of Oregon State University
InventorsStephen A. Johnston, William G. Dougherty
Life of this patent
Estimating the term…
Abstract
Methods for isolation and purification of recombinant proteins are described. Fusion proteins incorporating a cleavage site sensitive to proteolysis by a plant virus proteinase may be cleaved from carrier proteins to provide high yields of protein product. Methods employing a plant virus proteinase to cleave expressed fusion proteins are particularly suitable for obtaining proteolytically sensitive polypeptides in the presence of added cell protease inhibitors. Also disclosed are recombinant vectors useful for overproducing plant virus proteinases in a suitable host.
Claims20
A method of producing and isolating a recombinant polypeptide, comprising:
- transforming a yeast or gram-negative bacterial cell with a recombinant DNA molecule that comprises a first DNA encoding a matrix-binding protein, a second DNA encoding a tobacco etch virus NIa proteinase cleavage site, and a third DNA encoding a polypeptide;
- binding the expressed fusion protein to a matrix to a matrix that selectively binds the matrix-binding protein;
- and
- contacting matrix bound fusion protein with active, soluble recombinant 27 kDa tobacco etch virus NIa proteinase fused with a tag that selectively binds to an adsorbent material;
- wherein said proteinase selectively cleaves said cleavage site to release the recombinant polypeptide.
A method of overproducing a recombinant, active 27 kDa tobacco etch virus proteinase comprising:
- transforming a yeast or gram-negative bacterial cell with a recombinant vector which comprises a DNA encoding a 27 kDa tobacco etch virus proteinase linked with a DNA encoding a fusion partner polypeptide that selectively binds to an affinity matrix;
- and
- culturing the transformed cell to express a fusion protein that undergoes self-proteolysis to produce soluble, active 27 kDa NIa tobacco etch virus proteinase.
A method of overproducing recombinant tobacco etch virus 27 kDa NIa proteinase, comprising:
- transforming an E. coli or S. cerevisiae cell with a recombinant vector that includes a DNA encoding a matrix-binding protein, a DNA encoding a tobacco etch virus cleavage site, and a 27k DNA segment of tobacco etch virus proteinase NIa;
- and
- culturing the transformed cell to express a fusion protein that undergoes self-proteolysis at the tobacco etch virus NIa proteinase cleavage site to produce soluble, active 27 kD NIa tobacco etch virus proteinase.
A purified, active, recombinant tobacco etch virus 27 kDa proteinase NIa.
A recombinant 27 kDa tobacco etch virus proteinase NIa fused with alkaline phosphatase, .beta.-galactosidase, glutathione-S-transferase, or polyhistidine.
Dependencies are inferred from the claim text.