Production of bioactive porcine mutant myostatin propeptide/Fc fusion protein in Escherichia Coli
Skeletal muscle mass is negatively regulated by myostatin (MSTN), implying that MSTN inhibition would be a potential approach to increase skeletal muscle mass of meat producing animals. The activity of MSTN is suppressed by MSTN propeptide (MSTNPro), the N-terminal region of unprocessed MSTN that is cleaved off during post-translational MSTN processing. The objective of current study was to produce a mutant form of porcine MSTNPro fused to the Fc region of pig immunoglobulin G (pMSTNProM-Fc) in E. coli in order to examine its potential as an agent to enhance muscle mass in pigs. The pMSTNProM-Fc cDNA was constructed and cloned into pMAL-c5x vector downstream of the maltose-binding protein (MBP) gene, then was transformed and expressed in soluble forms in E. coli. For each L of cell culture at 4℃ for 7 days, about 13 mg of soluble MBP-pMSTNProM-Fc protein was purified by amylose-resin affinity chromatography. Further purification by protein A agarose affinity chromatography yielded about 0.64 mg/L culture of MBP-pMSTNProM-Fc protein. MBP-pMSTNProM-Fc inhibited MSTN bioactivity in a dosage-dependent manner in an in vitro gene reporter assay. The capacity of MBP-pMSTNProM-Fc to inhibit MSTN was comparable to those of MBP-pMSTNPro produced in E. coli and commercially-available murine MSTNPro produced in an eukaryotic system. Results from the current study show that Fc fusion to MBP-pMSTNPro does not affect the bioactivity of MBP-pMSTNPro, and the production of bioactive, mutant form of pig MSTN propeptide/Fc fusion protein is possible in E. coli.
Keywords: myostatin propeptide, pig, Fc fusion protein