Asparaginyl-tRNA 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 (50875 Da). Codon usage in the asparaginyl-tRNA synthetase gene (asnS) is similar to the characteristic usage in the genes for proteins from bacteria of the genus Thermus, and the G+C content in the third position of the codons is as high as 94%. The amino acid sequence of asparaginyl-tRNA synthetase from T. thermophilus shows high similarity with other bacterial asparaginyl-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 the enzyme were obtained from polyethylene glycol 6000 solutions by vapour diffusion techniques. The crystals diffract X-rays beyond 2.8 A.
Seignovert
L
L
EMBL Grenoble Outstation, France.
Härtlein
M
M
Leberman
R
R
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D000926:Anticodon; D001226:Aspartate-tRNA Ligase; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D003460:Crystallization; D017931:DNA Primers; D004926:Escherichia coli; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics; D014961:X-Ray Diffraction
D000926:Anticodon; D003062:Codon; D017931:DNA Primers; D010446:Peptide Fragments; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; D000604:Amino Acyl-tRNA Synthetases; D001226:Aspartate-tRNA Ligase; C029194:asparaginyl-tRNA synthetase