| Myostatin (MSTN), also known as growth and differentiation factor8(GDF-8),is a member of the transforming growth factor β (TGF-β) superfamily. Myostatin caninhibit the growth of skeletal muscle, so, it acts as a negative regulator of skeletalmuscle growth. Myostatin is a kind of secreted protein, mainly generated by theskeletal muscle and circulated in the blood as precursor protein which has nobiological activity. When the body needs, this precursor protein forms adisulfide-linked homodimer after twice protease digestion. myostatin inhibit skeletalmuscle growth in a concentration-dependent manner. Pig promyostatin wasconstituted by375amino acids:1~23constitute signal peptide,24~266constitutepropeptide,267~375constitute mature peptide. Firstly, promyostatin translocated tothe endoplasmic reticulum and removal of the signal peptide. Then, it was enzyme cutat the266amino acid site by metal protease to generate N-terminal propeptide andC-terminal mature peptide which was constituted by109amino acid residues.C-terminal forms a homodimer through nine disulfide bonds of the cysteine. Thehomodimer interact with its receptors which were on the cell membrane, then send thesignal incoming to cell nucleus through the mediated of three kinds of smad proteinand regulate target genes in order to regulate skeletal muscle fiber number and size.The76aspartic acid of mouse myostatin propeptide is the specific cutting site ofmetalloproteinases of bone morphogenetic proteins-1/tolloid (BMP-1/TLD) family.Myostatin propeptide was cleaved at here by protease and release the C-terminaldimer which was combined with propeptide by non-covalent bond. Then, C-terminaldimmer combines with its receptor and activating downstream signaling pathways.When the aspartate was mutated to alanine, propeptide still can combine withC-terminal dimmer and form an inactive latent complex, but this complex can resistthe specific enzyme cutting action of metalloproteinases of bone morphogeneticproteins-1/tolloid (BMP-1/TLD) family and inhibit the effect of myostatin on musclefiber and then led to the increase of skeletal muscle fiber number and size. The effectof mutated myostatin propeptide provides new research methods and ideas for curemuscle atrophy in medical science and improve animal muscle production in animalhusbandry. Therefore, it has good application prospect and economic value. In thisstudy, the total RNA was extracted from longissimus of3-month embryo ofTongcheng pig and the sequence of MSTN propeptide was amplified by RT-PCR.Then, site-directed mutagenesis was successfully converted Asp into Ala in the75 amino acid site in propeptide sequence. The myostatin propeptide and its mutatedform were subcloned into Prokaryotic expression vector pGEX-6p-1. The Prokaryoticexpression vectors of pGEX-ProM(SP-) and pGEX-ProM (SP-) D75A weresuccessfully constructed. Finally, the recombinant plasmids were expressed in E. coli(BL21) and induced with IPTG. The result shows that the expression product washighest when E.coli BL21transformed by pGEX-ProM(SP-) induced with0.6mmol/LIPTG for5hours, while， expression product was highest when E.coli BL21transformed by pGEX-ProM(SP-)-D75A induced with1.2mmol/L IPTG for6hours.The fusion protein of myostatin propeptide and its mutated form were obtained byGST-Trap system which the molecular weight is51KDa consist of26KDa GST tagand25KDa myostatin propeptide. The concentrations of ProM (-SP) and ProM (-SP)D75A were1.78mg/mL and1.21mg/mL after purify, respectively. this studyestablished an optimal condition for E. coli expression of the procine MSTNpropeptide and improved the production efficiency. Furthermore, the research will laya good foundation for studying the functions of porcine MSTN propeptide gene invivo and provide useful materials for the preparation of monoclonal antibody. |
PDF Full Text Request