Lee Sang Beum, Park Sung Kwon, Kim Yong Soo
Wide River Institute of Immunology, Seoul National University College of Medicine, Hongcheon, Gangwon-do, South Korea.
Department of Human Nutrition, Food and Animal Sciences, University of Hawaii at Manoa, Honolulu, HI, United States of America.
PLoS One. 2017 Apr 3;12(4):e0174956. doi: 10.1371/journal.pone.0174956. eCollection 2017.
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.
肌肉生长抑制素（MSTN）是骨骼肌生长的一种强效负调节因子。MSTN前肽（MSTNpro）通过与MSTN形成复合物来抑制MSTN与其受体的结合，这意味着MSTNpro可能是一种可用于促进产肉动物骨骼肌生长的有效物质。四种不同的截短形式的猪MSTNpro，其N端含有麦芽糖结合蛋白（MBP）作为融合伙伴，在大肠杆菌中表达，并通过亲和层析和凝胶过滤相结合的方法进行纯化。在体外基因报告试验中检测了这些蛋白质对MSTN的抑制能力。一种融合了MBP的截短MSTNpro，包含第42至175位残基（MBP-Pro42-175），表现出与全长序列MSTNpro相同的MSTN抑制效力。包含第42至115位残基（MBP-Pro42-115）或第42至98位残基（MBP-Pro42-98）的截短MSTNpro蛋白也表现出MSTN抑制能力，尽管其效力明显低于全长序列MSTNpro。在下拉试验中，MBP-Pro42-175、MBP-Pro42-115和MBP-Pro42-98证明了它们与MSTN的结合。通过与凝血因子Xa孵育，从截短的MSTNpro蛋白中去除MBP，以研究MBP对这些蛋白MSTN抑制能力的潜在作用。从MBP-Pro42-175和MBP-Pro42-98中去除MBP导致Pro42-175的MSTN抑制能力下降20倍，Pro42-98的MSTN抑制能力丧失，这表明作为融合伙伴的MBP增强了这些截短MSTNpro蛋白的MSTN抑制能力。总之，本研究表明，MBP是增强截短形式的MSTNpro蛋白的MSTN抑制效力的非常有用的融合伙伴，由第42至175位氨基酸残基组成的融合了MBP的猪MSTNpro足以维持完整的MSTN抑制能力。
