Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

中文翻译：

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通过可去除的骨架修饰方法化学合成膜蛋白。

化学合成可以产生带有专门设计的修饰（例如，磷酸化，同位素标记）的膜蛋白，而这些蛋白很难通过重组蛋白表达方法获得。所得均质修饰的合成膜蛋白是许多高级生物化学和生物物理研究的有价值的工具。该方案描述了通过天然化学连接通过跨膜肽段的缩合进行膜蛋白的化学合成。为避免由于跨膜肽在几乎所有溶剂中的溶解度差而遇到的常见问题，我们描述了一种通过可去除主链修饰（RBM）策略化学合成膜蛋白的有效程序。该协议的两个关键步骤是：（i）通过Fmoc（9-芴基甲氧羰基）固相肽合成将可溶Arg4标记的RBM基团安装在跨膜肽中的任何伯氨基酸上，以及（ii）全长序列的天然连接，然后去除通过TFA（三氟乙酸）混合物对RBM进行标记，从而得到天然蛋白质。RBM基团的安装是通过使用4-甲氧基-5-硝基水杨酰基酸酐通过还原胺化反应结合的活化的O-N酰基转移助剂实现的。Arg4-tag修饰的跨膜肽段的行为类似于水溶性肽，可促进其纯化，连接和质量表征。然后通过TFA（三氟乙酸）混合物去除RBM标签，得到天然蛋白。RBM基团的安装是通过使用4-甲氧基-5-硝基水杨酰基酸酐通过还原胺化反应结合的活化的O-N酰基转移助剂实现的。Arg4-tag修饰的跨膜肽段的行为类似于水溶性肽，可促进其纯化，连接和质量表征。然后通过TFA（三氟乙酸）混合物去除RBM标签，得到天然蛋白。RBM基团的安装是通过使用4-甲氧基-5-硝基水杨酰基酸酐通过还原胺化反应结合的活化的O-N酰基转移助剂实现的。Arg4-tag修饰的跨膜肽段的行为类似于水溶性肽，可促进其纯化，连接和质量表征。