Inhibition of fish myostatin activity by recombinant fish follistatin and myostatin prodomain: Potential implications for enhancing muscle growth in farmed fish

Myostatin (MSTN) is a negative regulator of skeletal muscle mass and has a potential application in aquaculture. Inactivation of MSTN results in significant muscle mass growth, a phenomenon known as “double muscling”. As part of our efforts to define ways in order to lower MSTN in fish, leading to enhanced muscle growth, we report here on the expression and purification of two potential binding proteins to fish MSTN: MSTN prodomain and follistatin (FST), both cloned from the marine fish, gilthead sea bream, Sparus aurata. MSTN prodomain was expressed in Escherichia coli, refolded and purified using anion-exchange chromatography. FST was stably expressed in CHO-K1 cells and partially purified from conditioned medium by affinity chromatography on Ni-NTA column. Secreted FST is glycosylated as evidenced from the reduction in its apparent molecular weight following treatment with Peptide N-glycosidase F seen on SDS-PAGE. Both MSTN prodomain and FST inhibited the biological activity of purified, cleaved and activated recombinant S. aurata MSTN in a dose-dependent manner, using the mammalian CAGA reporter gene assay. These results demonstrate that the inhibitory effect of MSTN prodomain and FST on MSTN activity, probably through binding, is conserved in fish. Moreover, these results offer an approach for lowering bioactive MSTN and thereby enhance muscle growth in fish important for aquaculture.