Biosynthesis and Genetic Encoding of Non-hydrolyzable Phosphoserine into Recombinant Proteins in Escherichia coli.
Biosynthesis and Genetic Encoding of Non-hydrolyzable Phosphoserine into Recombinant Proteins in Escherichia coli.
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DOI：
10.21769/bioprotoc.4861
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发表时间：
2023-11-05
期刊：
影响因子：
0.8
通讯作者：
Cooley, Richard B.
中科院分区：
文献类型：
--
作者：
Zhu, Phillip;Mehl, Ryan A.;Cooley, Richard B.
关键词：
虽然通过遗传密码扩增（GCE）技术在大肠杆菌中将磷酸丝氨酸（pSer）位点特异性翻译编码成蛋白质已经改变了我们研究磷酸蛋白结构和功能的能力，但重组磷酸蛋白可以在表达/纯化期间去磷酸化，并且它们暴露于细胞样环境（例如细胞裂解物）导致快速回复到非磷酸化形式。为了帮助克服这些挑战，我们开发了一个有效的和可扩展的E。coli GCE表达系统，能够将不可水解的磷酸丝氨酸（nhpSer）模拟物位点特异性掺入到目的蛋白中。这种nhpSer模拟物，其中磷酸丝氨酸的γ-氧被亚甲基（CH 2）基团取代，不受水解影响，并且即使当磷酸模拟物天冬氨酸和谷氨酸不受水解影响时，也重现磷酸丝氨酸功能。该表达系统的关键是链霉菌生物合成途径的共表达，该途径将中心代谢物磷酸烯醇丙酮酸转化为不可水解的磷酸丝氨酸（nhpSer）氨基酸，与培养基补充相比，通过增加nhpSer的生物利用度，其提供了> 40倍的表达产率改善，并实现了表达的可扩展性。该“PermaPhos”表达系统使用E. coli BL 21（DE 3）ΔserC菌株和三种质粒，它们表达（i）目的蛋白，（ii）用于在UAG琥珀终止密码子处翻译安装nhpSer的GCE机制，和（iii）链霉菌nhpSer生物合成途径。成功表达需要将所有三种质粒同时有效转化到表达宿主中，并且使用IPTG诱导所有组分的表达。在E.大肠杆菌的方法特别适用于从细胞裂解物或转染细胞中发现磷酸化依赖性蛋白质-蛋白质相互作用网络。 方案建立在Rogerson等人（2015）的nhpSer GCE系统上，但通过nhpSer生物合成途径使产量提高了40倍以上。·蛋白质表达使用标准Terrific Broth（TB）培养基，需要三天才能完成。·建议靶蛋白上的C-末端纯化标签，以避免过早截短的蛋白质与含全长nhpSer的蛋白质的共纯化。Phos标签凝胶电泳提供了一种确认准确nhpSer编码的方便方法，因为它可以区分非磷酸化的pSer和含nhpSer的变体。
While site-specific translational encoding of phosphoserine (pSer) into proteins in Escherichia coli via genetic code expansion (GCE) technologies has transformed our ability to study phospho-protein structure and function, recombinant phospho-proteins can be dephosphorylated during expression/purification, and their exposure to cellular-like environments such as cell lysates results in rapid reversion back to the non-phosphorylated form. To help overcome these challenges, we developed an efficient and scalable E. coli GCE expression system enabling site-specific incorporation of a non-hydrolyzable phosphoserine (nhpSer) mimic into proteins of interest. This nhpSer mimic, with the γ-oxygen of phosphoserine replaced by a methylene (CH2) group, is impervious to hydrolysis and recapitulates phosphoserine function even when phosphomimetics aspartate and glutamate do not. Key to this expression system is the co-expression of a Streptomyces biosynthetic pathway that converts the central metabolite phosphoenolpyruvate into non-hydrolyzable phosphoserine (nhpSer) amino acid, which provides a > 40-fold improvement in expression yields compared to media supplementation by increasing bioavailability of nhpSer and enables scalability of expressions. This “PermaPhos” expression system uses the E. coli BL21(DE3) ΔserC strain and three plasmids that express (i) the protein of interest, (ii) the GCE machinery for translational installation of nhpSer at UAG amber stop codons, and (iii) the Streptomyces nhpSer biosynthetic pathway. Successful expression requires efficient transformation of all three plasmids simultaneously into the expression host, and IPTG is used to induce expression of all components. Permanently phosphorylated proteins made in E. coli are particularly useful for discovering phosphorylation-dependent protein–protein interaction networks from cell lysates or transfected cells. Key features • Protocol builds on the nhpSer GCE system by Rogerson et al. (2015), but with a > 40-fold improvement in yields enabled by the nhpSer biosynthetic pathway. • Protein expression uses standard Terrific Broth (TB) media and requires three days to complete. • C-terminal purification tags on target protein are recommended to avoid co-purification of prematurely truncated protein with full-length nhpSer-containing protein. • Phos-tag gel electrophoresis provides a convenient method to confirm accurate nhpSer encoding, as it can distinguish between non-phosphorylated, pSer- and nhpSer-containing variants.