Genetic engineering of methionyl-tRNA synthetase: in vitro regeneration of an active synthetase by proteolytic cleavage of a methionyl-tRNA synthetase--beta-galactosidase chimeric protein.
The construction of a family of plasmids carrying derivatives of metG, the gene for E. coli methionyl-tRNA synthetase, is described. These plasmids allow expression of native or truncated forms of the enzyme and easy purification of the products. To facilitate the characterization of modified enzymes with very low catalytic activity, a specialized vector was constructed, in which metG was fused in frame with lacZ, the gene for beta-galactosidase. This plasmid expresses a methionyl-tRNA synthetase-beta-galactosidase chimeric protein, which is shown to carry the activities of both enzymes. This hybrid can be purified in a single step of affinity chromatography for beta-galactosidase. The methionyl-tRNA synthetase moiety can be regenerated by mild proteolysis, thus providing a simple method for purifying and studying mutated proteins.
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D002678:Chimera; D004926:Escherichia coli / Q000235:genetics; D005696:Galactosidases / Q000378:metabolism*; D005822:Genetic Vectors; D008718:Methionine-tRNA Ligase / Q000235:genetics*; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D001616:beta-Galactosidase / Q000378:metabolism*
D005696:Galactosidases; D001616:beta-Galactosidase; D010447:Peptide Hydrolases; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase