Glutamyl‐tRNA synthetase from Thermus thermophilus HB8

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Abstract
The gene for the Glu‐tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N‐terminal amino acid sequence of Glu‐tRNA synthetase. Nucleotide‐sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues (Mr 53901). Codon usage in the T. thermophilus Glu‐tRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G + C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T. thermophilus Glu‐tRNA synthetase showed high similarity with bacterial Glu‐tRNA synthetases (35–45% identity); the sequences of the binding sites for ATP and for the 3′ terminus of tRNAGlu are highly conserved. The Glu‐tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu‐tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three‐step column chromatography. Single crystals of T. thermophilus Glu‐tRNA synthetase were obtained from poly(ethylene glycol) 6000 solution by a vapor‐diffusion technique. The crystals diffract X‐rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P212121, with unit‐cell parameters of a= 8.64 nm, b= 8.86 nm and c= 8.49 nm.