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Overexpression and Purification of Thermus thermophilus Elongation Factors G, Tu, and Ts from Escherichia coli

1995; Elsevier BV; Volume: 6; Issue: 5 Linguagem: Inglês

10.1006/prep.1995.1084

1096-0279

Jutta Blank, Norbert Grillenbeck, Roland Kreutzer, Mathias Sprinzl,

Genomics and Phylogenetic Studies

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°C, respectively, and Sephacryl S200 gel permeation chromatography. For the purification of EF-Ts, a heat denaturation step is followed by DEAF-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-Cf 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.