Karakterisasi Protease dari Darah dan Pengklonan cDNA Faktor C dari Belangkas (Tachypleus gigas) Indonesia

Abstract

The horseshoe crab blood has some medical benefits, especially for endotoxin detection, which is an ability thought to be due to proteases’ role. Therefore, this study aimed to characterize proteases derived and to isolate factor C from horseshoe crab blood collected from Banyuasin Estuary, South Sumatra, Indonesia. With the blood sample, 1 EU/ mL of endotoxin was detected using the LAL test method, but a concentration of 0.5 EU/mL was not detected. Furthermore, the molecular mass was between 60-75 kDa and 25-35 kDa. It also had protease activity at a molecular mass below 60 kDa, while the highest activity was at temperatures 70 °C as well as pH 11. Besides, the enzyme’s activity was mostly inhibited in the presence of PMSF, EDTA, and SDS, but potentially increased by Tween-20 and cofactors such as Mg2+ and Ca2+. Based on the results, the enzymes contained in horseshoe crab blood belong to serine proteases. Light chain factor C fragment has been isolated from the blood of Tachypleus gigas (990 bp) and ligated to pGem-T-easy. The light chain sequence from T.gigas is similar to T. tridentatus sequence with a percent identity of 98.38%.