Thromb Haemost 1994; 71(03): 339-346
DOI: 10.1055/s-0038-1642440
Original Article
Schattauer GmbH Stuttgart

Refold and Characterization of Recombinant Tissue Factor Pathway Inhibitor Expressed in Escherichia coli

Authors

  • Judy A Diaz-Collier

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Mark O Palmier

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Kuniko K Kretzmer

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Bruce F Bishop

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Rodney G Combs

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Mark G Obukowicz

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Ronald B Frazier

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Gary S Bild

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • William D Joy

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Steven R Hill

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Kevin L Duffin

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Mark E Gustafson

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Kurt D Junger

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Roy W Grabner

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Gerald R Galluppi

    The Monsanto Corporate Research, Chesterfield, MO, USA
  • Tze-Chein Wun

    The Monsanto Corporate Research, Chesterfield, MO, USA
Further Information

Publication History

Received: 08 September 1993

Accepted after revision 16 September 1993

Publication Date:
06 July 2018 (online)

Preview

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.