title: Sequence, overproduction and crystallization of aspartyl-tRNA synthetase from Thermus thermophilus. Implications for the structure of prokaryotic aspartyl-tRNA synthetases.
abstract: The genes of aspartyl-tRNA synthetase (AspRS) from two Thermus thermophilus strain VK-1 and HB8, have been cloned and sequenced. Their nucleotidic sequences code for the same protein which displays the three characteristic motifs of class II aminoacyl-tRNA synthetases. This enzyme shows 50% identity with Escherichia coli AspRS, over the totality of the chain (580 amino acids). A comparison with the eukaryotic yeast cytoplasmic AspRS indicates the presence in the prokaryotic AspRS of an extra domain between motifs 2 and 3 much larger than in the eukaryotic ones. When its gene is under the control of the tac promoter of the expression vector pKK223-3, the protein is efficiently overexpressed as a thermostable protein in E. coli. It can be further purified to homogeneity using a heat treatment followed by a single anion exchange chromatography. Single crystals of the pure protein, diffracting at least to 2.2 A resolution (space group P2(1)2(1)2(1), a = 61.4 A, b = 156.1 A, c = 177.3 A) are routinely obtained. The same crystals have previously been described as crystals of threonyl-tRNA synthetase [1].
authors.affiliation: Laboratoire de Biochimie, URA 240 CNRS, Ecole Polytechnique, Palaiseau, France.
authors.forename: A
authors.initials: A
authors.lastname: Poterszman
authors.forename: P
authors.initials: P
authors.lastname: Plateau
authors.forename: D
authors.initials: D
authors.lastname: Moras
authors.forename: S
authors.initials: S
authors.lastname: Blanquet
authors.forename: M H
authors.initials: MH
authors.lastname: Mazauric
authors.forename: R
authors.initials: R
authors.lastname: Kreutzer
authors.forename: D
authors.initials: D
authors.lastname: Kern
mesh_terms: D000595:Amino Acid Sequence; D001226:Aspartate-tRNA Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D005798:Genes, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D009838:Oligodeoxyribonucleotides; D001226:Aspartate-tRNA Ligase