US Patent Number

6194169

Publication Date

2-27-2001

Abstract

Enhanced yields of the platelet-derived growth factor (PDGF) B-chain are obtained using the Pichia pastoris yeast system. Yields of both wild-type and mutant human proteins are enhanced when the yeast is transformed with a vector containing the yeast mating factor promoter fused to a sequence encoding the mature protein. The secreted wild-type protein is indistinguishable from mature 29-32 kDa protein isolated from human and Chinese hamster ovary (CHO) cells. An RKK→EEE mutant exhibited reduced association with the cell surface and accumulated in the culture medium as 29-32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK→EEE mutants of either the A- or B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor binding activities, each mutant retained full mitogenic activity when applied to cultured Swiss 3T3 cells. Circular dichroism spectrophotometric analysis of the RKK→EEE mutant revealed a secondary structure indistinguishable from the wild type. Yields of PDGF protein are increased many fold and in less time over previous methods.

Assignees

University of Kentucky Research Foundation, Lexington, KY (US)

Application Number

09/090,381

Filing Date

06/04/1998

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