Abstract
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.
MeSH terms
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Base Sequence
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Blood Coagulation / drug effects
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Carcinoma, Hepatocellular / chemistry
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DNA, Complementary / genetics
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Escherichia coli
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Factor Xa Inhibitors
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Gene Expression
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Genetic Vectors
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Humans
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Lipoproteins / biosynthesis*
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Lipoproteins / chemistry
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Lipoproteins / genetics
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Lipoproteins / isolation & purification
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Lipoproteins / pharmacology
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Liver Neoplasms / chemistry
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Mass Spectrometry
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Neoplasm Proteins / genetics
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Protein Folding
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Recombinant Fusion Proteins / biosynthesis*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / pharmacology
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Serine Proteinase Inhibitors / biosynthesis
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Serine Proteinase Inhibitors / chemistry
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Serine Proteinase Inhibitors / genetics
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Serine Proteinase Inhibitors / isolation & purification
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Serine Proteinase Inhibitors / pharmacology
Substances
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DNA, Complementary
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Factor Xa Inhibitors
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Lipoproteins
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Neoplasm Proteins
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Recombinant Fusion Proteins
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Serine Proteinase Inhibitors
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lipoprotein-associated coagulation inhibitor