Guerrero S A, Hecht H J, Hofmann B, Biebl H, Singh M
Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany.
Appl Microbiol Biotechnol. 2001 Sep;56(5-6):718-23. doi: 10.1007/s002530100690.
The amino acid analogue selenomethionine (SeMet) is shown to be efficiently incorporated into recombinant proteins expressed in Escherichia coli grown in a simple minimal medium without the addition of synthetic amino acids. Furthermore, satisfactory SeMet incorporation is obtained with a methionine-prototrophic strain transformed with commonly used vector systems. As examples, purified tryparedoxin 1 from Crithidia fasciculata, alkylhydroperoxide reductase (AhpC) from Mycobacterium marinum and the 16-kDa antigen from M. tuberculosis are shown to be efficiently labelled with SeMet, using the culture conditions and the host/vector systems described here. Enzymatic analysis reveals no differences between native and SeMet-labelled tryparedoxin 1 enzyme. Both proteins yield crystals under similar conditions. The culture conditions and host vector systems described greatly facilitate selenium-labelling of proteins for 3-D structure determination.
氨基酸类似物硒代蛋氨酸（SeMet）已被证明能有效地掺入在简单基本培养基中生长的大肠杆菌所表达的重组蛋白中，而无需添加合成氨基酸。此外，用常用载体系统转化的甲硫氨酸原养型菌株能实现令人满意的SeMet掺入。例如，利用本文所述的培养条件和宿主/载体系统，来自纤细无脊椎锥虫的纯化的锥虫硫氧还蛋白1、来自海分枝杆菌的烷基过氧化氢还原酶（AhpC）以及来自结核分枝杆菌的16 kDa抗原都被证明能有效地用SeMet标记。酶学分析表明，天然的和SeMet标记的锥虫硫氧还蛋白1酶之间没有差异。两种蛋白在相似条件下都能形成晶体。本文所述的培养条件和宿主载体系统极大地促进了用于三维结构测定的蛋白质的硒标记。
