High-level expression and single-step purification of leucyl-tRNA synthetase from Escherichia coli

A T7 promoter-based His(6)-tagging vector has been constructed that directs the synthesis in Escherichia coli of fusion proteins containing a stretch of six histidine residues at the N terminus. The vector allows overproducti on and single-step purification of His(6)-fusion protein by immobilized met al (Ni2+) chelate affinity chromatography. The gene encoding leucyl-tRNA sy nthetase (leuS) was cloned into this vector and expressed in high level. Th e specific activity of the synthetase in the crude extract of E. coli JM109 (DE3) transformant containing the His(6)-tagging vector with the gene leuS was approximately 110 times that of JM109(DE3) (the host strain without the vector). The overproduced His(6)-fusion leucyl-tRNA synthetase can be puri fied to homogeneity under native conditions within 2 h by one-step affinity chromatography with an overall yield of 55%. The His(6)-tag at the N termi nus of leucyl-tRNA synthetase did not affect its aminoacylation activity or the secondary structure. (C) 1999 Academic Press.