Methods and materials for increasing expression of recombinant polypeptides
AssigneeXoma Technology Ltd.
InventorArnold H. Horwitz
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Abstract
The present invention provides novel methods and materials for increasing the expression of recombinant polypeptides. Methods and materials of the invention allow increased expression of transcription units that include recombinant DNA sequences which encode polypeptides of interest. The present invention provides expression vectors which contain multiple copies of a transcription unit encoding a polypeptide of interest separated by at least one selective marker gene and methods for sequentially transforming or transfecting host cells with expression vectors to increase transcription unit dosage and expression.
Claims143
A method of producing a recombinant polypeptide comprising:
- (a) culturing cells which have been transformed or transfected with a vector or segment thereof comprising multiple copies of a transcription unit separated by at least one selective marker gene, wherein the transcription unit encodes a polypeptide, under selective conditions;
- and
- (b) expressing said polypeptide from the multiple copies of the transcription unit.
A method of producing a recombinant polypeptide from multiple copies of a transcription unit comprising the following steps:
- (a) transfecting or transforming cells with a vector or segment thereof comprising multiple copies of a transcription unit separated by at least one selective marker gene wherein the transcription unit encodes a polypeptide;
- (b) culturing cells from step (a) under selective conditions of at least one of the selective marker gene(s) thereby selecting for cells transfected or transformed with the vector or segment thereof;
- (c) optionally repeating steps (a) and (b) on the cells to sequentially transfect or transform the cells with an additional vector or segment thereof from step (a) wherein each additional vector or segment thereof comprises a different selective marker gene;
- and
- (d) expressing the polypeptide from the multiple copies of the transcription unit.
A vector or segment thereof comprising multiple copies of a transcription unit separated by at least one selective marker gene wherein the transcription unit encodes a polypeptide.
A method of producing a recombinant polypeptide comprising:
- (a) culturing cells which have been transformed or transfected with a vector or segment thereof comprising multiple copies of a transcription unit each separated by a selective marker gene, wherein the transcription unit encodes a polypeptide, under selective conditions;
- and
- (b) expressing said polypeptide from the multiple copies of the transcription unit.
The method of claim 73 or 74 wherein the cells have been transformed or transfected sequentially with an additional vector or segment thereof, and wherein each additional vector or segment thereof comprises a different selective marker gene.
A vector or segment thereof comprising multiple copies of a transcription unit each separated by a selective marker gene wherein the transcription unit encodes a polypeptide.
Dependencies are inferred from the claim text.