Semisynthesis and folding of the potassium channel KcsA
Semisynthesis and folding of the potassium channel KcsA
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DOI：
10.1021/ja0266722
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发表时间：
2002-08-07
影响因子：
15
通讯作者：
Muir, TW
中科院分区：
文献类型：
--
作者：
Valiyaveetil, FI;MacKinnon, R;Muir, TW
在这篇文章中，我们描述了钾通道KcsA的半合成。KcsA的截断形式，包括160个氨基酸蛋白的前125个氨基酸，通过表达蛋白连接合成。这种截断的形式对应于蛋白质的整个跨膜区域，类似于以前在KcsA蛋白的晶体学研究中使用的结构。连接反应采用与KcsA残基1-73对应的n端重组α -硫酯颗粒和与残基74-125对应的合成c端颗粒进行。采用优化后的Boc-SPPS技术完成了c肽的化学合成。采用谷胱甘肽- s -转移酶（GST）和GyrA蛋白的双融合策略，对n肽α -硫酯进行重组表达。融合蛋白以不溶性包涵体的形式表达，将融合蛋白重新折叠，然后依次切割，去除GST标签和内含蛋白，从而释放n肽α -硫酯。在化学结扎后，KcsA多肽被折叠成四聚体状态，并结合到脂质囊泡中。KcsA四聚体与agitoxin-2结合的能力证实了折叠态的正确性。据我们所知，这项工作代表了首次报道的多聚膜蛋白的半合成，并突出了天然化学连接和表达蛋白连接在整体膜蛋白（半）合成中的潜在应用。
In this contribution we describe the semisynthesis of the potassium channel, KcsA. A truncated form of KcsA, comprising the first 125 amino acids of the 160-amino acid protein, was synthesized using expressed protein ligation. This truncated form corresponds to the entire membrane-spanning region of the protein and is similar to the construct previously used in crystallographic studies on the KcsA protein. The ligation reaction was carried out using an N-terminal recombinant pepticle alpha-thioester, corresponding to residues 1-73 of KcsA, and a synthetic C-terminal pepticle corresponding to residues 74-125. Chemical synthesis of the C-peptide was accomplished by optimized Boc-SPPS techniques. A dual fusion strategy, involving glutathione-S-transferase (GST) and the GyrA intein, was developed for recombinant expression of the N-peptide alpha-thioester. The fusion protein, expressed in the insoluble form as inclusion bodies, was refolded and then cleaved successively to remove the GST tag and the intein, thereby releasing the N-peptide alpha-thioester. Following chemical ligation, the KcsA polypeptide was folded into the tetrameric state by incorporation into lipid vesicles. The correctness of the folded state was verified by the ability of the KcsA tetramer to bind to agitoxin-2. To our knowledge, this work represents the first reported semisynthesis of a polytopic membrane protein and highlights the potential application of native chemical ligation and expressed protein ligation for the (semi)synthesis of integral membrane proteins.