Identification, cloning, and expression of a functional phenylalanyl-tRNA synthetase (pheRS) from Staphylococcus aureus

Phenylalanyl-tRNA synthetase (pheRS) is unique among aminoacyl tRNA synthet ases in that it is a heterotetrameric enzyme composed of two alpha -subunit s and two larger beta -subunits. In prokaryotes, the alpha- and beta -subun its of pheRS are encoded by the genes pheS and pheT, respectively. In this report we describe the isolation of a DNA fragment (3.52 kb) containing the pheS and pheT genes from a Staphylococcus aureus (WCUH29) genomic DNA libr ary. Both genes, found as a part of transcriptional operon, were predicted to encode polypeptides which showed strong primary and structural similarit y to prokaryotic phenylalanyl-tRNA synthetase alpha- and beta -subunits. We describe the high-level overexpression and purification of recombinant S. aureuspheRS using pheS and pheT genes as part of an artificial operon in Es cherichia coli. For comparative analysis we also report a procedure for the purification of native pheRS from S. aureus (Oxford Strain) and demonstrat e that Michaelis-Menten parameters for both recombinant and native enzyme, at least for phenylalanine tRNA aminoacylation are comparable. (C) 2001 Aca demic Press.