Overproduction of Phenylalanyl-tRNA Synthetase fromThermus thermophilusHB8 inEscherichia coli

Abstract

The phenylalanyl-tRNA synthetase (FRS) fromThermus thermophiluswas overproduced inEscherichia coli.Three different promoter systems were used for the overexpression of thepheSTgenes: thetac, araB,and T7 promoters. Despite several attempts for improvement, the overproduction of the FRS was lower than that found with most of the otherT. thermophilusgenes. Nevertheless, enzyme amounts sufficient for biochemical and biophysical studies could be obtained more easily from the overproducingE. colithan fromT. thermophilus,since at least fivefold higher specific FRS activity was present in the overproducing cells than inT. thermophilus.Also, a simple purification procedure was established. After heat treatment at 70°C to remove thermolabileE. coliproteins, only three chromatographic steps, i.e., Q-Sepharose FF, hydroxyl apatite, and heparin–Sepharose chromatography, were necessary to obtain apparently homogeneous FRS. With a different plasmid construction we introduced six histidine residues at the N terminus of the α subunit. Thus, affinity chromatography on a nickel-chelate matrix can be used for the purification of FRS as well as for its mutant variants, which may be less stable than the native FRS and cannot be purified with heat treatment. We also cloned thepheSTgenes in a phagemid, which will enable mutagenesis studies and overexpression in a one-vector system without any subcloning steps.