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US 5,212,091 AGranted
Method of producing tissue factor pathway inhibitor
AssigneeMonsanto Company
InventorsJudy A. Diaz-Collier, Mark E. Gustafson, Tze-Chein Wun
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AssigneeMonsanto Company
InventorsJudy A. Diaz-Collier, Mark E. Gustafson, Tze-Chein Wun
Estimating the term…
A method is disclosed for the production of tissue factor pathway inhibitor (TFPI) which comprises expression of a non-glycosylated form of TFPI in E. coli as host and obtaining a highly active TFPI by in vitro refolding of the protein in which inclusion bodies isolated from the E. coli cells are subjected to stepwise purification comprising either series (A) or series (B) as follows: (A) (1) subjecting the inclusion bodies to sulfitolysis to form TFPI-S-sulfonate, (2) purifying TFPI-S-sulfonate by anion exchange chromatography, (3) refolding TFPI-S-sulfonate by disulfide interchange reaction, and (4) purifying active refolded TFPI by cation exchange chromatography, or (B) (1) subjecting the inclusion bodies to reduction with .beta.-mercaptoethanol in urea to form reduced TFPI, (2) purifying the reduced TFPI by cation exchange chromatography, (3) refolding reduced TFPI by disulfide interchange reaction in urea, and (4) purifying the active refolded TFPI by cation exchange chromatography.
A method for the production of a non-glycosylated form of tissue pathway inhibitor (SEQ ID No. 1) which comprises culturing under fermentation conditions sufficient to produce TFPI E. coli cells which have been transformed with a replicable expression vector containing the cDNA coding for said TFPI , harvesting the E. coli cells, isolating the inclusion bodies from the harvested E. coli cells and subjecting the inclusion bodies to a stepwise purification comprising either series (A) or series (B) as follows:
Dependencies are inferred from the claim text.