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Sequence, overproduction and crystallization of aspartyl‐tRNA synthetase from Thermus thermophilus
Author(s) -
Poterszman Arnaud,
Plateau Pierre,
Moras Dino,
Blanquef Sylvain,
Mazauric Marie-Hélène,
Kreutzer Roland,
Kern Daniel
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)81069-c
Subject(s) - thermus thermophilus , thermus , escherichia coli , transfer rna , biology , gene , biochemistry , enzyme , aminoacyl trna synthetase , amino acid , peptide sequence , microbiology and biotechnology , thermophile , rna
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 P2 1 2 1 2 1 , 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].