The Cloning, Expression And Purification Of MGF Gene

Human mechano growth factor (hMGF) is a basic polypeptide containing 110 amino acid residues. hMGF can active satellite cells, promote multiplication of cells and restrain differentiation. hMGF has visible effect on keeping the mass of local tissue and restoring the tissue, it has high potential of inducing the synthesize of local protein and preventing decease of cells.To obtain enough hMGF for basic or clinic research, we must choice an convenient method other than extraction from human materials (blood or tissue, eg). In this article, we synthesize the DNA sequence of hMGF and cloned in pMAL-p2x, pET-32a(+) and pET-3c vector by T vector transition. After confirmed the DNA sequence of pMAL-p2x-hMGF1, pET-32a(+)-hMGF1 and pET-3c-hMGF2 correctly, we transformed it into E.coli BL21 or BL21(DE3)plyss-star. After induced by IPTG, we found that pMAL-p2x is the best vector by SDS-PAGE. After the fermentation of pMAL-p2x-hMGF1/ BL21, we found that the fusion proteins are inclusion bodies when the bacteria broke by ultrasonic. The inclusion bodies were washed and dissolved by different urea concentration and then purified by Ni-NTA chromatography column. The obtained protein was renatured and purified gradually. At last, it was excised by EK enzyme and we gained the natural hMGF protein. After identified by SDS-PAGE, the purity of the obtained protein is above 90%.The results demonstrated that we had gained pMAL-p2x-hMGF1 successfully, and it could be expressed in inclusion bodies form at high level in BL21. After purification, renature and excision, we gained the natural hMGF protein. The pure protein could be used in biological active research, basic research and clinical practice.