Selenocysteine in native chemical ligation and expressed protein ligation
Selenocysteine in native chemical ligation and expressed protein ligation
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DOI：
10.1021/ja005885t
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发表时间：
2001-05-30
影响因子：
15
通讯作者：
Raines, RT
中科院分区：
文献类型：
--
作者：
Hondal, RJ;Nilsson, BL;Raines, RT
L-硒代半胱氨酸（Sec或U）被称为“第21种氨基酸”。1与20种常见氨基酸一样，硒代半胱氨酸在mRNA翻译过程中插入，并有自己的tRNASec和密码子UGA。该密码子也用作蛋白石终止密码子。将UGA密码子解码为硒代半胱氨酸密码子需要mRNA 3 '非翻译区中的特殊结构，称为硒代半胱氨酸插入序列（SECIS）元件。因为真核细胞和原核细胞使用不同的SECIS元件将UGA解码为硒代半胱氨酸，所以在原核生物中生产真核含硒代半胱氨酸的蛋白是有问题的。2在这里，我们描述了一个一般的半合成路线，含有硒代半胱氨酸的蛋白质。3，4在“天然化学连接”中，一个肽中的N-末端半胱氨酸残基的硫醇盐攻击另一个肽中的C-末端硫酯，最终在两个肽之间产生酰胺键（方案1）。5“表达蛋白连接”是通过使用重组DNA（rDNA）技术产生C-末端硫酯的延伸。6我们推断硒代半胱氨酸，像半胱氨酸一样，可以影响天然化学连接和表达的蛋白质连接，从而提供将硒代半胱氨酸掺入蛋白质的方法。我们使用AcGlySCH 2C（O）NHCH 3作为模型硫酯来测试在天然化学连接中使用硒代半胱氨酸的可行性。7在还原剂三-（2-羧乙基）膦（TCEP）存在下与胱氨酸（（CysOH）2）反应产生AcGly-CysOH以及一些（AcGlyCysOH）2。当在相同反应中使用硒代胱氨酸（（SecOH）2）时，产物为
L-Selenocysteine (Sec or U) has been called the “21st amino acid”. 1 Like the twenty common amino acids, selenocysteine is inserted during the translation of mRNA and has its own tRNASec and codon, UGA. This codon also serves as the opal stop codon. Decoding a UGA codon as one for selenocysteine requires a special structure in the 3′ untranslated region of the mRNA called a selenocysteine insertion sequence (SECIS) element. Because eukaryotic and prokaryotic cells use a different SECIS element to decode UGA as selenocysteine, the production of eukaryotic selenocysteine-containing proteins in prokaryotes is problematic. 2 Here, we describe a general semisynthetic route to proteins containing selenocysteine. 3, 4In “native chemical ligation”, the thiolate of an N-terminal cysteine residue in one peptide attacks a C-terminal thioester in another peptide to produce, ultimately, an amide bond between the two peptides (Scheme 1). 5 “Expressed protein ligation” is an extension in which the C-terminal thioester is produced by using recombinant DNA (rDNA) technology. 6 We reasoned that selenocysteine, like cysteine, could effect both native chemical ligation and expressed protein ligation, and thereby provide a means to incorporate selenocysteine into proteins. We used AcGlySCH2C (O) NHCH3 as a model thioester to test the feasibility of using selenocysteine in native chemical ligation. 7 Reaction with cystine ((CysOH) 2) in the presence of the reducing agent tris-(2-carboxyethyl) phosphine (TCEP) produced AcGly-CysOH, as well as some (AcGlyCysOH) 2. When selenocystine ((SecOH) 2) was used in the same reaction, the product was