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

Sang Beum Lee,Sung Kwon Park,Yong Soo Kim,Sabato D'Auria

doi:10.1371/journal.pone.0174956

Sang Beum Lee, Sung Kwon Park + Show 2 more

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https://doi.org/10.1371/journal.pone.0174956

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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.

Highlights

Since there is a possibility that the aggregated proteins may have little bioactivity, the aggregates were removed by gel filtration (Fig 2C), and gel filtration-purified maltose binding protein (MBP)-MSTN propeptide (MSTNpro) proteins were used in bioactivity examination
The current study shows that a MBP-fused pig MSTNpro consisting of amino acid residues 42–175 is sufficient to maintain the full MSTN-inhibitory capacity
Been unknown whether a fusion partner could play a role in modulating MSTN-inhibitory capacity of the truncated forms of MSTNpro, and the current results show that MBP fusion enhances MSTN-inhibitory capacity of the truncated forms of MSTNpro

Summary

Introduction

Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily and negatively regulates skeletal muscle growth and development with little effect on other tissues. Bioactivity of truncated forms of pig myostatin propeptide [1,2,3]. Similar to other TGF-β family member proteins, MSTN is translated as a precursor protein (preproMSTN) consisting of a signal sequence, an N-terminal propeptide domain (MSTNpro) and a C-terminal mature (active) domain [2, 4]. Upon entering into the endoplasmic reticulum, signal peptide is removed from preproMSTN, proMSTN forms a disulfide-linked homodimer and is proteolytically processed at a conserved Arg-Arg-X-Arg site by the furin family of proprotein convertases to generate MSTNpro and mature MSTN [5, 6]. Administration or overexpression of MSTNpro has been shown to enhance skeletal muscle growth in laboratory animal species [4, 5, 9,10,11,12,13,14], supporting the inhibitory role of MSTNpro on MSTN activity

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