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Refold and characterization of recombinant tissue factor pathway inhibitor expressed in Escherichia coli

Diaz-Collier, J.A.; Palmier, M.O.; Kretzmer, K.K.; Bishop, B.F.; Combs, R.G.; Obukowicz, M.G.; Frazier, R.B.; Bild, G.S.; Joy, W.D.; Hill, S.R.

Thrombosis and Haemostasis 71(3): 339-346

1994

ISSN/ISBN: 0340-6245
PMID: 8029799
Document Number: 438394
Human tissue factor pathway inhibitor (TFPI) was expressed in E. coli as a non-glycosylated protein with an additional alanine attached to the aminoterminus of the wild type molecule. High-level expression was obtained with pMON6875, a plasmid containing a tac promoter, Gene 10 leader 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 containing. TFPI were sulfitolyzed, purified by anion-exchange chromatography, refolded through a disulfide interchange reaction, and further fractionated by Mono S cation exchange chromatography. The Mono S resin resolved a peak of highly active TFPI from relatively inactive and possibly misfolded molecules. The E. coli TFPI was shown to be about two-fold more active, on a molar basis, than full-length human SK hepatoma TFPI in a tissue factor-induced clotting assay in human plasma.

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