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

Phenylalanyl-tRNA synthetase (pheRS) is unique among aminoacyl tRNA synthetases in that it is a heterotetrameric enzyme composed of two alpha-subunits and two larger beta-subunits. In prokaryotes, the alpha- and beta-subunits 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 library. Both genes, found as a part of transcriptional operon, were predicted to encode polypeptides which showed strong primary and structural similarity to prokaryotic phenylalanyl-tRNA synthetase alpha- and beta- subunits. We describe the high-level overexpression and purification of recombinant S. aureus pheRS using pheS and pheT genes as part of an artificial operon in Escherichia coli. For comparative analysis we also report a procedure for the purification of native pheRS from S. aureus (Oxford Strain) and demonstrate that Michaelis-Menten parameters for both recombinant and native enzyme, at least for phenylalanine tRNA aminoacylation are comparable.