Expression of human soluble tissue factor in yeast and enzymatic properties of its complex with factor VIIa.
The extracellular domain of human tissue factor (TF, amino acids 1-217) was expressed in Saccharomyces cerevisiae, using the inducible yeast acid phosphatase promoter and the yeast invertase signal sequence to direct its secretion into the culture broth. Two active soluble forms sTF alpha (high molecular weight form) and sTF beta (low molecular weight form) were purified, the yield being approximately 10 and 1 mg/liter of culture supernatant, respectively. sTF alpha had an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis and contained more than 200 residues of mannose/mol of protein. sTF beta had an apparent molecular mass of 37 kDa and contained 22 residues of mannose/mol of protein. N-Glycosidase F treatments of both rTFs reduced the apparent molecular mass to 35 kDa. The amino-terminal sequences and amino acid compositions of sTF alpha and sTF beta were consistent with those deduced from the cDNA sequence, thereby indicating that the difference in molecular mass is caused by heterogeneity of oligosaccharide structures. Of these recombinant TFs, sTF beta enhanced factor VIIa-amidolytic activity 40-fold toward the chromogenic substrate and 147-fold toward the fluorogenic substrate, affecting mainly the kcat value. The enhancement was comparable with that of TF purified from human placenta. The TF-mediated enhancement of factor VIIa-amidolytic activity was inhibited by heparin-activated antithrombin III, forming a high molecular weight complex. As treatment of sTF beta with denaturants such as guanidine hydrochloride or urea led to a biphasic loss of the activity, the extracellular domain of TF probably consists of two discrete domains. This expression system provides a significant amount of the extracellular domain of TF so that studies of interactions with factor VII are feasible.
Shigematsu
Y
Y
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Miyata
T
T
Higashi
S
S
Miki
T
T
Sadler
J E
JE
Iwanaga
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D015942:Factor VIIa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity; D013925:Thromboplastin / Q000235:genetics* / Q000378:metabolism
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D004247:DNA; D013925:Thromboplastin; D015942:Factor VIIa