RENATURATION AND PURIFICATION OF HUMAN TISSUE FACTOR PATHWAY INHIBITOR EXPRESSED IN RECOMBINANT ESCHERICHIA-COLI

Tissue factor pathway inhibitor is an inhibitor of the extrinsic coagu lation pathway. Evaluation of the pharmacological effects of tissue fa ctor pathway inhibitor in animal models has been limited by the high c ost and low availability of mammalian tissue culture produced protein. In order to circumvent this obstacle, a 277-amino-acid nonglycosylate d tissue factor pathway inhibitor variant possessing an N-terminal ala nine was expressed in recombinant E. coli using the tac promoter expre ssion system. High-level expression in recombinant E. coli resulted in the accumulation of alatissue factor pathway inhibitor in inclusion b odies. Active protein was produced by solubilization of the inclusion bodies in 8 M urea, purification of the full-length molecule by cation exchange chromatography, and renaturation in 6 M urea. Fractionation of crude refold mixtures using cation exchange chromatography yielded a purified nonglycosylated tissue factor pathway inhibitor possessing in vitro prothrombin time activity comparable to inhibitor purified fr om mammalian cell lines. (C) 1994 Academic Press, Inc.