Cloning,Expression,Purification And Characterization Of Human Macrophage Metalloelastase Catalytic Domain Gene

Objective In this experiment we cloned 507 bp cDNA fragment coding for human macrophage metalloelastase catalytic domain (HMEcd) and constructed a prokaryotic expression plasmid and expressed it in E. coli.Then HMEcd protein was purified and renatured, which can be used in the studies on tumor intervention with HME.Methods HMEcd cDNA was amplified by reverse transcriptional polymerase chain reaction(RT-PCR) from tissue of gastric cancer and identified by 1% agarose gel electrophoresis. The fragment was inserted into pMD18-T vector and constructed pMD18-T-HMEcd plasmid. The plasmid was transformed into E. coli DH5αand amplified in the bacterium. Then we extracted pMD18-T-HMEcd plasmid from E. coli DH5αand identified the recombinant plasmid by double digestion with restriction enzymes BamH I and Xho I and sequencing. HMEcd cDNA cutted from pMD18-T-HMEcd was subcloned into pET-28a(+) vector. The recombinant pET-28a(+)-HMEcd was identified with the same method for pMD18-T-HMEcd and transformed into E. coli BL21(DE3) and induced by IPTG. The production of expression was analyzed by SDS-PAGE and detected by Western-blot. The fusion protein was purified with His Bind Collumn and refolded by dialysis and assaied after concentration. The enzymatic activity of recombinant protein was confirmed by gelatin zymography and cleavage of type I collagen in vitro.Results HMEcd cDNA was cloned. The pMD18-T-HMEcd and pET-28a(+)-HMEcd plasmid were conducted and confirmed. SDS-PAGE and Western-blot demonstrated HMEcd protein was expressed in E. coli BL-21(DE3). The recombinant protein occupied approximately 18.92% of total bacterial protein. HMEcd protein was purified with a purity of over 98% and demonstrated the enzymatic activity.Conclusions We obtained HMEcd cDNA and prokaryotic expressive plasmid perfectly.The recombinant HMEcd fusion protein was expressed successfully. We established the purification and refolding scheme through the experiments. Which laid the foundation for successive studies.