Evaluating the Effect of Protein A Signal Peptide on Extracellular Expression of Recombinant Hirudin in E.Coli

Abstract         Background and Objective: Hirudin is an anticoagulant polypeptide secreted from the salivary glands of leeches. Recombinant hirudin is a strong anticoagulant agent in arterial and venous thrombosis. The aim of this study was to evaluate the effect of inserting protein A signal peptide sequence of pEZZ18 plasmid on expression and secretion of the recombinant hirudin in E.coli.        Methods: the synthetic hirudin gene was amplified by PCR using specific primers. First, the gene was purified and cloned into PTG19-T cloning vector, and then it was subcloned into pEZZ18 expression vector by SalI / SacI enzymatic digestion and finally transformed into E.coli JM107. After the expression of recombinant hirudin protein, different cellular fractions were isolated and analyzed on SDS-PAGE and further confirmed by Western blotting.          Results: PCR product (522 bp) was first subcloned into the T-Vector (replicating vector) and then successfully subcloned into the pEZZ18 (expression vector). Cloning and subclonig were confirmed by enzymatic digestion and Colony PCR. After the expression and isolation of fractions, the presence of hirudin (about 29 kDa) in different cell fractions due to the effects of signal peptide was observed in SDS-PAGE and finally confirmed by Western blotting.        Conclusion: The gene of anticoagulant hirudin protein (desirudin) was cloned into the pEZZ18 vector containing Protein A signal peptide sequence and later transformed into E.coli JM107. The recombinant hirudin protein expression in the extracellular space was approved.         Keywords: Hirudin; Desirudin; Protein Sorting Signals.