Refold and characterization of recombinant tissue factor pathway inhibitor expressed in Escherichia coli.
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.
Monsanto Corporate Research, Chesterfield, MO 63198.
J A
JA
Diaz-Collier
M O
MO
Palmier
K K
KK
Kretzmer
B F
BF
Bishop
R G
RG
Combs
M G
MG
Obukowicz
R B
RB
Frazier
G S
GS
Bild
W D
WD
Joy
S R
SR
Hill
D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli; D065427:Factor Xa Inhibitors; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008074:Lipoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D008113:Liver Neoplasms / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D018076:DNA, Complementary; D065427:Factor Xa Inhibitors; D008074:Lipoproteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; C051928:lipoprotein-associated coagulation inhibitor