Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut.
Department of Biosciences, Rice University, Houston, Texas.
Curr Protoc. 2021 Feb;1(2):e54. doi: 10.1002/cpz1.54.
Selenoproteins contain the 21st amino acid, selenocysteine. Selenocysteine is the only amino acid that is synthesized on its cognate tRNA, and it is inserted at specific recoded UGA stop codons via a complex translation system. Although highly similar to cysteine, selenocysteine has unique properties, including a stronger nucleophilic ability and lower reduction potential. Efforts to site-specifically incorporate selenocysteine to create recombinant selenoproteins involve a recoded UAG stop codon and expression of the necessary selenocysteine translation machinery. This article presents a protocol for expressing and purifying selenoproteins in Escherichia coli. © 2021 Wiley Periodicals LLC. Basic Protocol: Recombinant selenoprotein production in E. coli using a rewired translation system.
硒蛋白含有第 21 种氨基酸——硒代半胱氨酸。硒代半胱氨酸是唯一在其相应的 tRNA 上合成的氨基酸，它通过一个复杂的翻译系统插入到特定的被重编码的 UGA 终止密码子上。尽管硒代半胱氨酸与半胱氨酸高度相似，但它具有独特的性质，包括更强的亲核能力和更低的还原电位。为了定点掺入硒代半胱氨酸来创建重组硒蛋白，涉及到重编码的 UAG 终止密码子和表达必要的硒代半胱氨酸翻译机制。本文介绍了在大肠杆菌中表达和纯化硒蛋白的方案。© 2021 威利父子公司。基本方案：使用重布线翻译系统在大肠杆菌中生产重组硒蛋白。
