|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1994 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1993 : ¥1,400,000 (Direct Cost : ¥1,400,000)
In the previous study involving a yeast expression system, a high molecular mass extracellular domain of human tissue factor (denoted as sTFalpha) with a high content of mannose residues was produced in abundance and 37 kDa sTFbeta was obtained in a low yield (Shigematsu et al.(1992) J.Biol.Chem.267,21329-21337). To obtain sTFbeta in a high yield, we constructed four kinds of mutant sTF with partial or total replacement of the N-potential glycosylation Asn residues with Ala, and expressed them in yeast. We found that the yield of the beta form of the Asn137-to-Ala mutant (designated as sTFbetaNNA) was three-fold higher (3mg/liter) than that of the wild type, suggesting that the replacement of one of the three potential N-glycosylation Asn residues with Ala could be a good way to minimize the addition of mannose repeats. High recovery of the 37 kDa sTFbeta now opened a way for the analysis of three-dimensional structure. Since it has been reported that calcium ions are required for the
effective hydrolysis of peptidyl substrates by the factor VIIa-sTF complex, it is believed to be essential for the expression of full protease activity. Here, we report the enzymatic characterization of a factor VIIa-sTFbetaNNA complex cross-linked with a homobifunctional reagent, bis (sulfosuccinimidyl) suberate. The factor VIIa-sTFbetaNNA complex cross-linked in the presence of 5 mM calcium ions or 50 mM EDTA was purified. The cross-linked complex did not show factor X activation in the presence of phospholipids. However, it showed essentially the same activity toward peptidyl substrates as before cross-linking, even in the presence of EDTA.The kinetic constants of the cross-linked complexes in the presence of 5 mM calcium and 50 mM EDTA were similar, indicating that once factor VIIa was cross-linked with sTF,calcium ions were required no more for its activity. Therefore, calcium ions are not required for full catalytic activity of the factor VIIa-sTF complex toward synthetic substrates once the bi-molecular complex has formed.