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

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
› Author Affiliations
Further Information

Publication History

Received: 08 September 1993

Accepted after revision 16 September 1993

Publication Date:
06 July 2018 (online)

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.