Renaturation and purification of human tissue factor pathway inhibitor expressed in recombinant E. coli.
Tissue factor pathway inhibitor is an inhibitor of the extrinsic coagulation pathway. Evaluation of the pharmacological effects of tissue factor pathway inhibitor in animal models has been limited by the high cost and low availability of mammalian tissue culture produced protein. In order to circumvent this obstacle, a 277-amino-acid nonglycosylated tissue factor pathway inhibitor variant possessing an N-terminal alanine was expressed in recombinant E. coli using the tac promoter expression system. High-level expression in recombinant E. coli resulted in the accumulation of ala-tissue factor pathway inhibitor in inclusion bodies. Active protein was produced by solubilization of the inclusion bodies in 8 M urea, purification of the full-length molecule by cation exchange chromatography, and renaturation in 6 M urea. Fractionation of crude refold mixtures using cation exchange chromatography yielded a purified nonglycosylated tissue factor pathway inhibitor possessing in vitro prothrombin time activity comparable to inhibitor purified from mammalian cell lines.
Gustafson
M E
ME
Monsanto Corporate Research, Chesterfield, Missouri 63198.
Junger
K D
KD
Wun
T C
TC
Foy
B A
BA
Diaz-Collier
J A
JA
Welsch
D J
DJ
Obukowicz
M G
MG
Bishop
B F
BF
Bild
G S
GS
Leimgruber
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D008074:Lipoproteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D008297:Male; D008322:Mammals; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D011517:Prothrombin Time; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology
D008074:Lipoproteins; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor