REFOLD AND CHARACTERIZATION OF RECOMBINANT TISSUE FACTOR PATHWAY INHIBITOR EXPRESSED IN ESCHERICHIA-COLI

Human tissue factor pathway inhibitor (TFPI) was expressed in E. coli as a non-glycosylated protein with an additional alanine attached to t he aminoterminus of the wild type molecule. High-level expression was obtained with pMON6875, a plasmid containing a tac promoter, Gene 10 l eader from bacteriophage T7, methionine-alanine-TFPI coding sequence, and the p22 transcriptional terminator. In this system, TFPI accounted for about 5-10% of the total cell protein. The inclusion bodies conta ining TFPI were sulfitolyzed, purified by anion-exchange chromatograph y, refolded through a disulfide interchange reaction, and further frac tionated by Mono S cation exchange chromatography. The Mono S resin re solved a peak of highly active TFPI from relatively inactive and possi bly misfolded molecules. The E. coli TFPI was shown to be about two-fo ld more active, on a molar basis, than full-length human SK hepatoma T FPI in a tissue factor-induced clotting assay in human plasma.