Mutation of the gene encoding γ-sarcoglycan (SGCG), an integral membrane protein responsible for maintaining the integrity of the muscle cell sarcolemma, results in Limb-Girdle Muscular Dystrophy (LGMD), a congenital disease with no current treatment options. This member of the sarcoglycan glycoprotein family is a vital component of the Dystrophin Complex, which together facilitate normal muscle function. However, very little is known about the structure and dynamics of these proteins, and of membrane glycoproteins in general. This is due to a number of factors, including their complexity, heterogeneity and highly-specific native environments. The expression, purification, and structural study of membrane proteins is further impeded by their hydrophobic nature and consequent propensity to aggregate in aqueous solutions. Here, we report the first successful expression and purification of milligram quantities of full-length recombinant SGCG, utilizing fusion protein-guided overexpression to inclusion bodies in Escherichia coli. Purification of SGCG from the fusion protein, TrpΔLE, was facilitated using chemical cleavage. Cleavage products were then isolated by size-exclusion chromatography. Successful purification of the protein was confirmed using SDS-PAGE and mass spectroscopy. Finally, solution nuclear magnetic resonance spectroscopy of uniformly 15N-labeled SGCG in detergent environments was performed, yielding the first spectra of the full-length membrane glycoprotein, SGCG. These results represent the initial structural studies of SGCG, laying the foundation for further investigation on the interaction and dynamics of other integral membrane proteins. More specifically, this data allows for opportunities in the future for enhanced treatment modalities and cures for LGMD.

中文翻译：

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全长人整合膜蛋白γ-糖聚糖的表达，纯化和结构分析。

编码γ-糖聚糖（SGCG）（负责维持肌肉细胞肉瘤的完整性的完整膜蛋白）的基因的突变会导致肢带型肌营养不良症（LGMD），这是一种先天性疾病，目前尚无治疗方法。肌聚糖糖蛋白家族的这个成员是肌营养不良蛋白复合物的重要组成部分，它们共同促进正常的肌肉功能。但是，对这些蛋白质以及一般膜糖蛋白的结构和动力学了解甚少。这是由于许多因素引起的，包括它们的复杂性，异质性和高度特定的本机环境。膜蛋白的疏水性及其在水溶液中聚集的倾向进一步阻碍了膜蛋白的表达，纯化和结构研究。这里，我们报道了首次成功的表达和纯化的毫克量的全长重组SGCG，利用融合蛋白引导的过表达来包涵体在大肠杆菌中。使用化学裂解可促进从融合蛋白TrpΔLE中纯化SGCG。然后通过尺寸排阻色谱分离切割产物。使用SDS-PAGE和质谱证实了蛋白质的成功纯化。最后，在去污剂环境中对15N标记的SGCG进行了溶液核磁共振波谱分析，得出了全长膜糖蛋白SGCG的第一张光谱图。这些结果代表了SGCG的初步结构研究，为进一步研究其他完整膜蛋白的相互作用和动力学奠定了基础。