Department of Human Nutrition, Food and Animal Sciences, University of Hawaii at Manoa, 1955 East West Rd, Honolulu, HI, 96826, USA.
Appl Microbiol Biotechnol. 2013 Oct;97(19):8517-27. doi: 10.1007/s00253-013-5134-0. Epub 2013 Aug 3.
Myostatin (MSTN) is a potent negative regulator of skeletal muscle mass. The activity of MSTN is suppressed by MSTN propeptide (MSTNPro), the N-terminal part of unprocessed MSTN that is cleaved off during posttranslational MSTN processing. Easy availability of MSTNPro would help to investigate the potential of the protein as an agent to enhance muscle growth in agricultural animal species. Thus, this study was designed to produce bioactive wild-type porcine MSTN propeptide (pMSTNProW) and its mutated form at the BMP-1/TLD proteolytic cleavage site (pMSTNProM) in Escherichia coli. The pMSTNProW and pMSTNProM genes were separately cloned into pMAL-c5X vector downstream of the maltose-binding protein (MBP) gene and were transformed and expressed in soluble forms in E. coli. For each milliliter of cell culture, about 40 μg of soluble MBP-pMSTNProW and MBP-pMSTNProM proteins were purified by amylose resin affinity chromatography. Further purification by anion exchange chromatography of the affinity-purified fractions yielded about 10 μg/mL culture of MBP-pMSTNProW and MBP-pMSTNProM proteins. Factor Xa protease cleaved the fusion partner MBP from MBP-pMSTNPro proteins, and approximately 4.2 μg of pMSTNProW and pMSTNProM proteins were purified per milliliter of culture. MBP-pMSTNProM was resistant to digestion by BMP-1 metalloproteinase, while MBP-pMSTNProW was cleaved into two fragments by BMP-1. Both MBP-pMSTNProW and MBP-pMSTNProM demonstrated their MSTN binding affinities in a pulldown assay. In an in vitro gene reporter assay, both proteins inhibited MSTN bioactivity without a significant difference in their inhibitory capacities, indicating that the cell culture-based gene reporter assay has limitation in detecting the true in vivo biological potencies of mutant forms of MSTNPro proteins at the BMP-1/TLD cleavage site. Current results show that a high-level production of bioactive porcine MSTNpro is possible in E. coli, and it remains to be investigated whether the administration of the MSTNpro can improve skeletal muscle growth in pigs via suppression of MSTN activity in vivo.
肌肉生长抑制素 (MSTN) 是一种强效的骨骼肌肉质量负调节剂。MSTN 的活性受到 MSTN 前肽 (MSTNPro) 的抑制，MSTNPro 是未经加工的 MSTN 的 N 端部分，在翻译后加工过程中被切割。MSTNPro 的易得性将有助于研究该蛋白作为增强农业动物物种肌肉生长的潜在药物。因此，本研究旨在在大肠杆菌中生产生物活性的野生型猪 MSTN 前肽 (pMSTNProW) 及其在 BMP-1/TLD 蛋白水解切割位点突变的形式 (pMSTNProM)。pMSTNProW 和 pMSTNProM 基因分别克隆到 pMAL-c5X 载体下游的麦芽糖结合蛋白 (MBP) 基因中，并在大肠杆菌中以可溶性形式转化和表达。对于每毫升细胞培养物，约 40μg 的可溶性 MBP-pMSTNProW 和 MBP-pMSTNProM 蛋白通过麦芽糊精树脂亲和层析纯化。亲和纯化级分的阴离子交换层析进一步纯化，得到约 10μg/mL 培养物的 MBP-pMSTNProW 和 MBP-pMSTNProM 蛋白。因子 Xa 蛋白酶将融合伴侣 MBP 从 MBP-pMSTNPro 蛋白中切割下来，每毫升培养物中可纯化出约 4.2μg 的 pMSTNProW 和 pMSTNProM 蛋白。MBP-pMSTNProM 抵抗 BMP-1 金属蛋白酶的消化，而 MBP-pMSTNProW 被 BMP-1 切割成两个片段。MBP-pMSTNProW 和 MBP-pMSTNProM 在下拉测定中均显示出其与 MSTN 的结合亲和力。在体外基因报告测定中，两种蛋白均抑制 MSTN 活性，但其抑制能力无显著差异，表明基于细胞培养的基因报告测定在检测 BMP-1/TLD 切割位点 MSTNPro 蛋白突变形式的真正体内生物学效力方面存在局限性。目前的结果表明，在大肠杆菌中可以高水平生产生物活性的猪 MSTNpro，仍有待研究 MSTNpro 的给药是否可以通过抑制体内 MSTN 活性来改善猪的骨骼肌肉生长。
