Efficient expression of tyrosine-sulfated proteins in E. coli using an expanded genetic code.
Efficient expression of tyrosine-sulfated proteins in E. coli using an expanded genetic code.
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DOI：
10.1038/nprot.2009.188
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发表时间：
2009
期刊：
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
--
作者：
酪氨酸硫酸化是一种重要的翻译后修饰，发生在高等真核生物中，并参与细胞间通讯、病毒进入和粘附。我们描述了一种在大肠杆菌中异源表达选择性酪氨酸硫酸化蛋白的方法。大肠杆菌通过使用扩展的遗传密码子，该扩展的遗传密码子响应于琥珀无义密码子TAG而保守地插入磺基酪氨酸。该过程所需的组分（磺基酪氨酸特异性正交氨酰-tRNA合成酶及其识别琥珀密码子的同源正交tRNA）在质粒pSUPAR 6-L3-3SY上编码;其用途以及磺基酪氨酸的简单化学合成沿着在本方案中概述。具体地，突变目的蛋白质的基因，使得对应于酪氨酸硫酸盐的所需位置的密码子是TAG。将含有该基因的表达载体与pSUPAR 6-L3-3SY共转化到合适的E.大肠杆菌菌株允许以高效率和保真度过表达位点特异性硫酸化蛋白。所得蛋白质在TAG密码子指定的任何位置含有酪氨酸硫酸盐，使得该方法比竞争方法（例如体外酶促硫酸化、化学硫酸化和肽合成）显著更简单且更通用。一旦克隆了合适的表达载体，我们的方案应该允许在不到一周的时间内生产所需的硫酸化蛋白。
Tyrosine sulfation is an important posttranslational modification that occurs in higher eukaryotes and is involved in cell-cell communication, viral entry, and adhesion. We describe a protocol for the heterologous expression of selectively tyrosine-sulfated proteins in E. coli through the use of an expanded genetic code that cotranslationally inserts sulfotyrosine in response to the amber nonsense codon, TAG. The components required for this process, an orthogonal aminoacyl-tRNA synthetase specific for sulfotyrosine and its cognate orthogonal tRNA that recognizes the amber codon, are encoded on the plasmid pSUPAR6-L3-3SY; and their use, along with a simple chemical synthesis of sulfotyrosine, is outlined in this protocol. Specifically, the gene for a protein of interest is mutated such that the codon corresponding to the desired location of tyrosine-sulfate is TAG. Cotransformation of an expression vector containing this gene and pSUPAR6-L3-3SY into an appropriate E. coli strain allows the overexpression of the site-specifically sulfated protein with high efficiency and fidelity. The resulting protein contains tyrosine-sulfate at any location specified by a TAG codon, making this method significantly simpler and more versatile than competing methods such as in vitro enzymatic sulfation, chemical sulfation, and peptide synthesis. Once the proper expression vectors are cloned, our protocol should allow the production of the desired sulfated proteins in less than one week.
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