SEQUENCE, OVERPRODUCTION AND CRYSTALLIZATION OF ASPARTYL-TRANSFER RNA-SYNTHETASE FROM THERMUS-THERMOPHILUS - IMPLICATIONS FOR THE STRUCTUREOF PROKARYOTIC ASPARTYL-TRANSFER RNA-SYNTHETASES

The genes of aspartyl-tRNA synthetase (AspRS) from two Thermus thermop hilus strains VK-1 and HB8, have been cloned and sequenced. Their nucl eotidic sequences code for the same protein which displays the three c haracteristic motifs of class II aminoacyl-tRNA synthetases. This enzy me shows 50% identity with Escherichia coli AspRS, over the totality o f the chain (580 amino acids). A comparison with the eukaryotic yeast cytoplasmic AspRS indicates the presence in the prokaryotic AspRS of a n extra domain between motifs 2 and 3 much larger than in the eukaryot ic 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 ho mogeneity using a heat treatment followed by a single anion exchange c hromatography. Single crystals of the pure protein, diffracting at lea st to 2.2 A resolution (space group P2(1)2(1)2(1), a = 61.4 angstrom, b = 156.1 angstrom, c = 177.3 angstrom) are routinely obtained. The sa me crystals have previously been described as crystals of threonyl-tRN A synthetase [1].