CDNA cloning and expression of earthworm fibrinolytic enzyme component A

Abstract

Earthworm fibrinolytic enzyme component A (EFEa), a protein with dual fibrinolytic activity, is one of the major therapeutically important earthworm fibrinolytic enzyme components. The cDNA fragment encoded the mature protein was cloned from earthworm (Eisenia fetida) by the RT-PCR technique. The deduced amino acid sequence of the EFE component A shows high homology with some members of serine proteases trypsin family, and the amino acid residues constituting the active sites are conserved in the EFEa as compared with the other proteins of the trypsin family. The cDNA fragment was subcloned into the expression vector pQE31 and pMAL-c2X ofE. coli. The resulting expression plasmids, pQE-efea and pMAL-efea, were used to transform theE. coli strain M15. Recombinant protein bands corresponding with calculated molecular weights were induced. The induced His6-EFEa fusion protein with pQE-efea was accumulated into inclusion body, while the induced MBP-EFEa fusion protein with pMAL-efea was soluble and showed fibrinolytic activities.