Details

Autor(en) / Beteiligte

• 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

Titel

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

Ist Teil von

• Thrombosis and haemostasis, 1994-03, Vol.71 (3), p.339-346

Ort / Verlag

Germany

Erscheinungsjahr

1994

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.