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DOI: 10.1055/s-0038-1642440
Refold and Characterization of Recombinant Tissue Factor Pathway Inhibitor Expressed in Escherichia coli
Publikationsverlauf
Received: 08. September 1993
Accepted after revision 16. September 1993
Publikationsdatum:
06. Juli 2018 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/199403/lookinside/thumbnails/10-1055-s-0038-1642440-1.jpg)
Summary
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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