The Overproduction of Leucyl-tRNA Synthetase in E. coli and Its Purification

The 3.2 kb gene (leuS) encoding for leucyl-tRNA synthetase (LeuRS) has been cloned from E.coli K-12, and overexpressed 35 times more than that in the host strain TG1. In order to further increase the production of LeuRS, two types of leuS with different length of the 3' flanking region: leuS1 has an additional 130 bp over that of leuS2 were ligated into pKK-233-2 and pTrc-99B, respectively. In E. coli TG1 transformant harboring the recombination plasmid pTrc-99B with leuS2, the yield of LeuRS was 135 times that in TG1 strain. The purified enzyme that showed one band on SDS-PAGE was obtained after two steps of column chromatography. During the construction of plasmid, a substitute of G for C was introduced at position of base 4 in the coding region of leuS, so that Gln2 of LeuRS was changed to Glu. This enzyme was designated LeuRS2E. The kinetic constants of LeuRS2E showed that the substitution has no effect on the enzyme activity and could be used in the studies of LeuRS as the native enzyme.