title: 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.
authors.affiliation: 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.
authors.forename: Sang Beum
authors.initials: SB
authors.lastname: Lee
authors.affiliation: National Institute of Animal Science, RDA, Suwon, South Korea.
authors.forename: Sung Kwon
authors.initials: SK
authors.lastname: Park
authors.affiliation: Department of Human Nutrition, Food and Animal Sciences, University of Hawaii at Manoa, Honolulu, HI, United States of America.
authors.forename: Yong Soo
authors.identifier: http://orcid.org/0000-0001-6648-8430
authors.initials: YS
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D008460:Meat / Q000032:analysis; D024510:Muscle Development / Q000235:genetics / Q000502:physiology; D018482:Muscle, Skeletal / Q000254:growth & development; D055435:Myostatin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D013552:Swine
chemical_list: D058619:Maltose-Binding Proteins; D055435:Myostatin; D011993:Recombinant Fusion Proteins