Characterization of human tissue factor pathway inhibitor variants expressed in Saccharomyces cerevisiae.
Human tissue factor pathway inhibitor (TFPI) and three derivatives with deletions of: 1) the complete COOH-terminal third of the polypeptide including the third Kunitz domain, 2) the third Kunitz domain alone, or 3) the penultimate basic COOH-terminal region alone were expressed in yeast as secreted products. High expression yield was obtained only with the derivative that lacked both the third Kunitz domain and the penultimate COOH tail (TFPI1-161). The purified short form was heterogeneously glycosylated with a high mannose glycan. The specific activities of the different mutant polypeptides toward FXa.tissue factor.FVIIa in a chromogenic assay were similar to that of TFPI expressed in baby hamster kidney cells, suggesting that correct folding takes place in yeast and that neither the third Kunitz domain nor the COOH-terminal region is required for this activity. However, in a clotting assay the anticoagulant activities of yeast-produced TFPI and the shortened derivative TFPI1-161 were about 5- and 50-fold lower, respectively, than for full-length TFPI from mammalian cells. Clotting assays with purified short form TFPI showed that it acted mainly via inhibition of FVIIa.tissue factor rather than FXa. The anticoagulant activity of short form TFPI was comparable with that of high affinity antibodies toward tissue factor.
Novo Nordisk A/S, Novo Alle, Bagsvaerd, Denmark.
J G
JG
Petersen
G
G
Meyn
J S
JS
Rasmussen
J
J
Petersen
S E
SE
Bjørn
I
I
Jonassen
L
L
Christiansen
O
O
Nordfang
D000595:Amino Acid Sequence; D000925:Anticoagulants / Q000494:pharmacology; D002241:Carbohydrates / Q000032:analysis; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005167:Factor VII / Q000037:antagonists & inhibitors*; D006801:Humans; D008074:Lipoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D000925:Anticoagulants; D002241:Carbohydrates; D008074:Lipoproteins; C051928:lipoprotein-associated coagulation inhibitor; D005167:Factor VII