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

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Abstract
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].