ASPARAGINYL-TRANSFER-RNA SYNTHETASE FROM THERMUS-THERMOPHILUS HB8 SEQUENCE OF THE GENE AND CRYSTALLIZATION OF THE ENZYME EXPRESSED IN ESCHERICHIA-COLI

The gene for the asparaginyl-tRNA synthetase, a class IIb enzyme, from the extreme thermophile Thermus thermophilus HB8 has been cloned and sequenced. Sequence analysis revealed an open reading frame that codes for a protein of 438 amino acid residues (50 875 Da). Codon usage in the asparaginyl-tRNA synthetase gene (asnS) is similar to the characte ristic usage in the genes for proteins from bacteria of the genus Ther mus, and the G+C content in the third position of the codons is as hig h as 94 %. The amino acid sequence of asparaginyl-tRNA synthetase from T. thermophilus shows high similarity with other bacterial asparaginy l-tRNA synthetase sequences (30-55 % identity). By expression of the T . thermophilus asnS gene in Escherichia coli, the thermostable enzyme was overproduced and purified to homogeneity by heat treatment and two chromatography steps. The protein obtained is remarkably thermostable and retains 50 % of its initial tRNA aminoacylation activity after 1 h of incubation at 90 degrees C or 21 h at 85 degrees C. Crystals of t he enzyme were obtained from polyethylene glycol 6000 solutions by vap our diffusion techniques. The crystals diffract X-rays beyond 2.8 Angs trom.