Blank J, Grillenbeck N W, Kreutzer R, Sprinzl M
Lehrstuhl für Biochemie, Universität Bayreuth, Germany.
Protein Expr Purif. 1995 Oct;6(5):637-45. doi: 10.1006/prep.1995.1084.
The translation elongation factors G (EF-G), Tu (EF-Tu), and Ts (EF-Ts) from the extreme thermophilic bacterium Thermus thermophilus were overproduced in Escherichia coli. The fus gene coding for EF-G and the tufA gene coding for EF-Tu were expressed under the control of a tac promoter, whereas EF-Ts was overproduced with the T7 RNA polymerase system. A detailed description for the purification of the three elongation factors from E. coli is presented. EF-G and EF-Tu are isolated by Q-Sepharose FF chromatography, heat treatment at 65 or 60 degrees C, respectively, and Sephacryl S200 gel permeation chromatography. For the purification of EF-Ts, a heat denaturation step is followed by DEAE-cellulose chromatography and a cation exchange EMD-SO-3 650 column. The overproduced factors show the same properties as those purified from T. thermophilus. As the crystal structures of T. thermophilus EF-Tu and EF-G have been solved recently, many questions concerning the function of particular residues or domains arise, which may be best addressed by studying the in vitro behavior and structure of altered recombinant constructs. The methods presented here should facilitate such studies.
嗜热栖热菌的翻译延伸因子G（EF-G）、Tu（EF-Tu）和Ts（EF-Ts）在大肠杆菌中过量表达。编码EF-G的fus基因和编码EF-Tu的tufA基因在tac启动子的控制下表达，而EF-Ts则通过T7 RNA聚合酶系统过量表达。本文详细描述了从大肠杆菌中纯化这三种延伸因子的方法。EF-G和EF-Tu通过Q-Sepharose FF色谱法、分别在65或60℃下热处理以及Sephacryl S200凝胶渗透色谱法进行分离。对于EF-Ts的纯化，在热变性步骤之后进行DEAE-纤维素色谱法和阳离子交换EMD-SO-3 650柱层析。过量表达的因子表现出与从嗜热栖热菌中纯化的因子相同的性质。由于嗜热栖热菌EF-Tu和EF-G的晶体结构最近已得到解析，许多关于特定残基或结构域功能的问题随之出现，而这些问题最好通过研究改变后的重组构建体的体外行为和结构来解决。本文介绍的方法应有助于此类研究。
