Molecular Cloning, Sequencing, And Expression Of CDNA Encoding Serine Protease With Fibrinolytic Activity From Clamworm

Objective Cloning and expression a new serine protease with fibrinolytic activity from cDNA library established from clamworm digestive tract epithelial cells by RACE technology and detecting its bio-activity using artificial fibrin plates. Methods Extracted clamworm digestive tract epithelial cells total RNA by Trizol reagent, obtained its cDNA library and screening this library by a specific primer then got the positive clone.Sequenced this clone and predicted it by EXPASY Protemics tools. The results indicated this protein shows similarity to mammalian serine proteases and has the conserved catalytic amino acid residues. Cloned it into GST fusion protein expression vector pGEX4T-2, expressed under the IPTG induce. SDS-PAGE electrophoresis showed it is a 37KD fusion protein, purified this protein by thrombin cutting and examined its activity by artificial fibrin plates. Results The molecular weight of this protein was 37 000D and mainly expressed as inclusion body in Escherichia coli. Purified protein weighed 9 000D. Artificial fibrin plates test demonstrated that this purified protein presented fibrinolytic activity. Conclusion The prokaryotic expression protein can be used as a potential drug for thrombosis in clinical.