Sequence, overproduction and crystallization of aspartyl‐tRNA synthetase from Thermus thermophilus

FEBS Letters

Poterszman, Arnaud⁴  Plateau, Pierre⁴  Kern, Daniel³  Blanquef, Sylvain⁴  Kreutzer, Roland²  Mazauric, Marie-Hélène³  Moras, Dino¹ 

[1] Laboratoire de Biologie Structurale, UPR 9004 CNRS, 15 rue René Descartes, 67084 Strasbourg Cédex, France;Laboratorium für Biochemie, Universität Bayreuth, Universitätsstrasse 30, 8580 Bayreuth, Germany;Unité ‘Structure des Macromolécules Biologiques et Mecanismes de Reconnaissance’, 15 rue René Descartes, 67084 Strasbourg Cédex, France;Laboratoire de Biochimie, URA 240 CNRS, École Polytechnique, 91128 Palaiseau Cédex, France

关键词: Aminoacyl-tRNA synthetase;    Aspartyl-tRNA synthetase;    Thermus thermophilus. Crystallization;    aaRS;    aminoacyl-tRNA synthetase;    AsnRS;    AspRS;    GlnRS;    LysRS;    MetRS;    ThrRS and TyrRS;    asparaginyl-;    aspartyl-;    glutaminyl-;    lysyl-;    methionyl-;    threonyl- and tyrosyl-tRNA synthetase;    respectively;    ec;    Escherichia coli;    tt;    Thermus thermophilus;    y;    yeast cytoplasmic;    ym;    yeast mitochondrial;    IPTG;    isopropyl-β-thiogalactopyranoside;    bp;    basepair(s);    kb;    kilobasepair(s);   

DOI  :  10.1016/0014-5793(93)81069-C

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The genes of aspartyl-tRNA synthetase (AspRS) from two Thermus thermophilus strains 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 Å resolution (space group P2₁2₁2₁, a = 61.4 Å, b = 156.1 Å, c = 177.3 Å) are routinely obtained. The same crystals have previously been described as crystals of threonyl-tRNA synthetase [1].

【 授权许可】

Unknown