Protein Ligation and Labeling Enabled by a C-Terminal Tetracysteine Tag
Protein Ligation and Labeling Enabled by a C-Terminal Tetracysteine Tag
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C 端四半胱氨酸标签实现蛋白质连接和标记
DOI：
10.1002/anie.202115377
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通讯作者：
He Chunmao
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作者：
Mo Zeyuan;Lin Shaomin;Chen Wentao;He Chunmao
S-氰基化肽的肼解提供了一种获得蛋白质 α-酰肼的替代方法，α-酰肼是天然化学连接 (NCL) 中使用的关键试剂，无需任何内含肽或酶的帮助。然而，目前使用的非选择性 S-氰基化不允许蛋白质中除了切割位点的半胱氨酸之外再存在其他半胱氨酸。在此，我们报告了通过将四半胱氨酸标签融合到目标蛋白的 C 末端而实现的区域选择性 S-氰基化和肼解策略。我们将其称为四半胱氨酸蛋白连接 (TCEPL)。虽然具有高度选择性，但该策略适用于表达为包涵体的蛋白质，铁硫蛋白红氧还蛋白的有效半合成以及含有 207 个氨基酸残基的基质金属蛋白酶-14 (MMP-14) 的催化和铰链结构域证明了这一点。此外，TCEPL 策略被用于使用带有多种官能团的氨基试剂来标记蛋白质 C 末端，证明了其多功能性和通用性。
The hydrazinolysis of S‐cyanylated peptide provides an alternative way to afford protein α‐hydrazide, a key reagent used in native chemical ligation (NCL), without the aid of any inteins or enzymes. The currently used non‐selective S‐cyanylation, however, allows no other cysteine in the protein besides the one at the cleavage site. Herein, we report a regioselective S‐cyanylation and hydrazinolysis strategy achieved via the fusion of a tetracysteine tag to the C‐terminal of the protein of interest. We term it tetracysteine enabled protein ligation (TCEPL). While highly selective, the strategy is applicable for proteins expressed as inclusion bodies, and this was showcased by the efficient semi‐synthesis of an iron‐sulfur protein rubredoxin and the catalytic and hinge domains of matrix metalloprotease‐14 (MMP‐14) containing 207 amino acid residues. Furthermore, the TCEPL strategy was exploited for protein C‐terminal labeling with amino reagents bearing a variety of functional groups, demonstrating its versatility and generality.