Prokaryotic Expressionand Purification Of MMP9-PEX And Research Of Its Renaturation

ObjectiveThe noneatalytic carboxyl-terminal hemopexin domain(PEX) of matrix metalloproteinase, as an endogenous angiogenesis inhibitor, plays a domaint role in inhibiting neoplasms invasion and hematogenous metastasis. To optimize the prokaryotic expression, purification and renaturation conditions for the hight level expression of functional human carboxyl-terminal hemopexin domain(PEX) of matrix metalloproteinase(MMP9).MethodsWe extracted mRNA from normal humen liver through Trizol method, MMP9-PEX gene was amplified from mRNA of liver tisue by RT-PCR.The RT-PCR products and PET-His were simultaneously digested by Nhe I and Hindâ…¢,then purified,ligased,transformated and identified to construct the recombinant vector PET-is-MMP9-PEX. The expression vector,PET his MP9-PEX, was transformed into E.coli BL21(DE3) induced by IPTG and the expression level of PEX was investigeted in various culture conditions, Cytoplasmic Inclusions was clearaged by guanidine hydrochloride, the recombinant protein was purifiedby by affinity chromatography and the enzymatic activities of MMP-9-PEX were detected by gelatin zymography.ResultsFirstly we extracted mRNA from normal human liver successfully through Trizol method,the result of RT-PCR products in the garose gel elctrophrosis show that there had positive strap,which matched the expected size.Through digested and sequenced,the inserted DNA sequence was the gene of gene of MMP9-PEXwas transformed into E.coli BL21(DE3) induced by IPTG and the expression level of PEX was investigeted in various culture conditions,The purity of renatured protein is above 95% and the renaturation rate is 36.86%.ConclusionsWe extracted mRNA from normal human liver successfully through Trizol method,and gain the cDNA of MMP-9-PEX by RT-PCR properly,the recombinant vector PET-his-MMP9-PEX has successfully constructed,and expressed after being transfected into BL21(DE3). The prokaryotic expression, purification and renaturation conditiones for the expression of functional human hemopexin domain of matrix metalloproteinase have been optimized and the functional protein of MMP9-PEX has been prepared for further functional study.