Maltose binding protein-fusion enhances the bioactivity of truncated forms of pig myostatin propeptide produced in E. coli

Abstract

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth. MSTN propeptide (MSTNpro) inhibits MSTN binding to its receptor through complex formation with MSTN, implying that MSTNpro can be a useful agent to improve skeletal muscle growth in meat-producing animals. Four different truncated forms of pig MSTNpro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in E. coli, and purified by the combination of affinity chromatography and gel filtration. The MSTN-inhibitory capacities of these proteins were examined in an in vitro gene reporter assay. A MBP-fused, truncated MSTNpro containing residues 42–175 (MBP-Pro42-175) exhibited the same MSTN-inhibitory potency as the full sequence MSTNpro. Truncated MSTNpro proteins containing either residues 42–115 (MBP-Pro42-115) or 42–98 (MBP-Pro42-98) also exhibited MSTN-inhibitory capacity even though the potencies were significantly lower than that of full sequence MSTNpro. In pull-down assays, MBP-Pro42-175, MBP-Pro42-115, and MBP-Pro42-98 demonstrated their binding to MSTN. MBP was removed from the truncated MSTNpro proteins by incubation with factor Xa to examine the potential role of MBP on MSTN-inhibitory capacity of those proteins. Removal of MBP from MBP-Pro42-175 and MBP-Pro42-98 resulted in 20-fold decrease in MSTN-inhibitory capacity of Pro42-175 and abolition of MSTN-inhibitory capacity of Pro42-98, indicating that MBP as fusion partner enhanced the MSTN-inhibitory capacity of those truncated MSTNpro proteins. In summary, this study shows that MBP is a very useful fusion partner in enhancing MSTN-inhibitory potency of truncated forms of MSTNpro proteins, and MBP-fused pig MSTNpro consisting of amino acid residues 42–175 is sufficient to maintain the full MSTN-inhibitory capacity.

Identification of the minimum region of flatfish myostatin propeptide (Pep45-65) for myostatin inhibition and its potential to enhance muscle growth and performance in animals

“…Recent studies have shown that synthetic peptides containing 29 or 24 amino acids from the mouse MSTNpro were sufficient to suppress MSTN activity in vitro and to increase muscle mass in mice of muscular dystrophy model [12, 13]. We also have shown that truncated forms of pig and flatfish MSTNpro with an N-terminal fusion of maltose binding protein (MBP) had the MSTN-inhibitory capacity not different from the full sequence mouse MSTNpro [35, 36]. Notably, the size of MSTNpro region of flatfish (residues 45–80) was much smaller than that of pig (residues 42–175) for full MSTN-inhibitory capacity, suggesting that the large sequence variation between fish and mammalian MSTNpro affects the stability of MSTNpro and MSTN complex.…”

“…Furthermore, intramuscular injection of the truncated MSTNpro peptides increased muscle mass in Duchenne and caveolin 3-deficient limb-girdle muscular dystrophy model mice [12, 13]. In our study, maltose binding protein (MBP)-fused pig MSTNpro region containing residues 42–175 exhibited full MSTN-inhibitory potency [35]. Similarly, flatfish MSTNpro region containing residues 45–80 showed MSTN-inhibitory potency comparable to that of full sequence mouse MSTNpro [36], indicating that MBP fused, truncated MSTNpro peptides would be potential agents to treat muscle wasting conditions.…”

Screening and identification of cyprinid herpesvirus 2 (CyHV-2) ORF55-interacting proteins by phage display

“…According to the preliminary analysis of physicochemical parameters, the ORF55 protein was unstable and not highly hydrophilic (Table 1 ), indicating that the final purified protein may not be highly soluble and/or easily degraded. Based on these analyses, the pMAL-c5X vector was selected for gene cloning because MBP is known to be highly soluble [ 41 ] and can enhance the biological activity of fused target proteins [ 42 ]. We successfully cloned the ORF55 gene into the pMAL-c5X vector, and expressed and purified the MBP-ORF55 fusion protein.…”

Determination of Complete Sequence Mutation of Myostatin Gene in Fast- and Slow-Growing Chicken

“…The results of this study indicated that there were no significant differences in gene expression and amino acid sequences of myostatin between fastand slow-growing groups of chickens. Allegedly, the physiological function of myostatin in not only affected by the myostatin gene expression but also affected by other molecules, i.e TGF-β1 (Zhu et al 2007), decorin (Zhu et al 2007), follistatin and follistatin associated genes (Lee and McPherron 2001), myostatin propeptide (Haq et al 2013;Lee et al 2016Lee et al , 2017, Bone Morphogenetic Protein-1 (Lee 2010), Tolloid (Lee 2010), and Tolloid-like-1 and 2 (Lee 2010).…”