Overproduction of the Thermus thermophilus elongation factor Tu in Escherichia coli

Abstract

The elongation factor Tu (EF-Tu) encoded by the tuf1 gene of the extreme thermophilic bacterium Thermus thermophilus HB8 was expressed under control of the tac promoter from the recombinant plasmid pEFTu-10 in Escherichia coli. Thermophilic EF-Tu-GDP, which amounts to as much as 35% of the cellular protein content, was separated from the E coli EF-Tu·GDP by thermal denaturation at 60°C. The overproduced E coli-born T thermophilus EF-Tu was characterized by: i) recognition through T thermophilus anti-EF-Tu antibodies; ii) analysis of the peptides obtained by cyanogen bromide cleavage; iii) thermostability; iv) guanine nucleotide binding activity in the absence and the presence of elongation factor Ts; and v) ternary complex formation with phenylalanyl-tRNA Phe and GTP.