Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology
Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology
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DOI：
10.1073/pnas.96.18.10068
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发表时间：
1999-08-31
影响因子：
11.1
通讯作者：
Dawson, PE
中科院分区：
文献类型：
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作者：
Hackeng, TM;Griffin, JH;Dawson, PE
蛋白质的全化学合成对于增加我们对蛋白质功能的分子基础的理解具有巨大的潜力。引入天然化学连接技术，将未受保护的肽连接到半胱氨酸残基旁，极大地促进了中等大小蛋白质的合成。我们描述了一种简单的方法，使我们能够快速分析X- cys连接位点的天然化学连接策略的兼容性，其中X是20种天然存在的氨基酸中的任何一种。简化的方法避免了特定氨基酸硫酯连接或c端硫氨酸肽烷基化的必要性。实验使用基质辅助激光解吸电离质谱分析lyrax - c末端硫酯肽与肽曲柄的组合连接表明，所有20个氨基酸都适合连接，Val， lie和Pro代表不太有利的选择，因为连接速度慢。为了说明该方法的实用性，两个124-aa蛋白通过三步四段式连接人工合成，得到一个完全活性的人分泌磷脂酶AZ和一个催化无活性的类似物。由于方法的简化，使设计的灵活性与通用性相结合，扩大了化学蛋白质合成的适用性和通用性。
The total chemical synthesis of proteins has great potential for increasing our understanding of the molecular basis of protein function. The introduction of native chemical ligation techniques to join unprotected peptides next to a cysteine residue has greatly facilitated the synthesis of proteins of moderate size. Were, we describe a straightforward methodology that has enabled us to rapidly analyze the compatibility of the native chemical ligation strategy for X-Cys ligation sites, where X is any of the 20 naturally occurring amino acids. The simplified methodology avoids the necessity of specific amino acid thioester linkers or alkylation of C-terminal thioacid peptides. Experiments using matrix-assisted laser-desorption ionization MS analysis of combinatorial ligations of LYRAX-C-terminal thioester peptides to the peptide CRANK show that all 20 amino acids are suitable for ligation, with Val, lie, and Pro representing less favorable choices because of slow ligation rates. To illustrate the method's utility, two 124-aa proteins were manually synthesized by using a three-step, four-piece ligation to yield a fully active human secretory phospholipase AZ and a catalytically inactive analog. The combination of flexibility in design with general access because of simplified methodology broadens the applicability and versatility of chemical protein synthesis.