Purification and characterization of recombinant protease with thrombolytic activity of Perinereis aibuhitensis Grube / 中国海洋药物

ABSTRACT

Objective To express and purify recombinant protease with thrombolysis activity of Perinereis aibuhitensis Grube and study on its characterization.Methods pMAL-PPA was in- troduced into E.coli DH5?to construct engineering bacteria and overexpression of the prote- ase of fused with maltose binding protein(MBP-PPA)was achieved with IPTG induction. The fusion protein was purified by affinity chromatography on amylose-resin column fol- lowed by chromatography on a DEAE-sepharose FF column.PPA cut with Factor Xa was assayed using casein plates supplied with plasminogen.Results Engineering bacteria express- ing maltose binding protein-thrombolytic protease of P.aibuhitensis were constructed and overexpression of MBP-PPA was achieved with IPTG induction.A recombinant fusion pro- tein of 51kD was purified,and PPA cut down from the fusion protein had a plasminogen-acti- rating activity.The protease showed a good thermal stability with an optimal pH of 8.0. This enzyme was also relatively stable in a pH range of 6.0～9.0 and still active after stored in organic solvents for 20d.Conclusion PPA was verified as a plasminogen activator,and might be a new thrombolytic medicine in the future.