Cloning,Expression And Purification Of MMP-2 CD In Sika Deer

Objective: Realization of in vitro expression of sika deer matrix metalloproteinase 2(Catalytic domain of matrix metalloprotease-2,cdMMP-2),study of the structural function of cdMMP-2 and product development and application.Methods: The catalytic domain of matrix metalloproteinase-2 cDNA was amplified by RT-PCR,and then the gene fragment was inserted into the recombinant prokaryotic expression vector pET30 a by Nde I and Not I.By bioinformatics analysis,the recombinant expression plasmid was transformed into the host bacteria E.coli BL21,the target protein was induced by IPTG,using metal chelate chromatography and dialysis methode purificate and refold the target protein firstly;then the activity of the target protein was detected by enzymatic degradation substrate method.Results: 1.There is a His tag at the C-terminal of the recombinant protein.This recombinant protein contains 377 amino acid,and has a predicted MW of 42.04 kDa and a theoretical isoelectric point of 4.95;2.By transforming BL21 competent cells and IPTG induction.,the target protein was successfully expressed,Its optimal IPTG induction concentration and optimal induction time is 0.6mM and 5h;3.SDS-polyacrylamide electrophoresis and immunoblot analysis showed that the target protein was mainly expressed in inclusion bodies.The protein was purified by metal chelate chromatography,SDS-PAGE result was a single;4.After column dialysis,the refolded cdMMP-14 is obtained,using the method of Bradford protein concentration protein concentration is2.0 mg/mL;6.cdMMP-14 has collagen hydrolysis activity of enzyme was detected by the method of enzymatic degradation substrate.Conclusion: This study obtained high purity,high yield,and activity MMP-2 by constructing prokaryotic expression system.