Construction and Characterization of the Recombinant Prokaryotic Vector pGEX-5X-3/MMP-11

Objective To construct the recombinant prokaryotic expression vector pGEX-5X-3/MMP-11 encoding the N-terminal region of matrix metalloproteinase 11 (MMP-11), and to obtain the fusion MMP-11 protein. Methods cDNA coding for the N-terminal region of MMP-11 was amplified from normal human endometrium by RT-PCR. The amplified fragments were then inserted into the expression vector pGEX-5X-3. Sequence of the insert was analyzed by digestion of the recombinant plasmid pGEX-5X-3/MMP-11 with restriction endonuclease and followed by sequencing. Recombinant plasmid was then transfected into E.coli DH5α and the positive clones were subsequently selected. GST-MMP-11 fusion protein was obtained by inducing the E.coli cells with β-D-thiogalactopyranoside. Results The MMP-11 cDNA fragment amplified by RT-PCR was found to be accurately inserted into pGEX-5X-3 vector. SDS-PAGE analysis revealed that the fusion protein had an apparent molecular weight of 4 550.Conclusion The recombinant prokaryotic expression vector pGEX-5X-3/MMP-11 was successfully constructed, and a 4 550 GST-MMP-11 fusion protein was obtained. The production of MMP-11 protein would facilitate the future development of anti-MMP-11 antibody for the purpose of cancer diagnosis and for the treatment of malignant diseases.