An insertion mutant of LeuRS with 116 amino acid residues has full activity

Escherichia coli leucyl-tRNA synthetase (LeuRS) belongs to class I aminoacyl-tRNA synthetases. It consists of 860 amino acid residues and catalyzes the leucylation of tRNA^leu. An insertion of its 253-368 peptide fragment between 368 to 369 in CP1 domain of this enzyme was shown to maintain the activity of the enzyme, and the insertion mutant was named as LeuRS-C. Because the insertion mutant of LeuRS was sensitive to operation of the purification, a plasmid containing the gene encoding LeuRS with His₆-tag at its N-terminus was constructed to facilitate the purification of His₆-LeuRS-C through one-step affinity chromatography on Ni-NTA column. The purified His₆-LeuRS-C had full activity as the native LeuRS with His-tag at the N-terminus (His₆-LeuRS), although the mutant enzyme had an insertion of 116 amino acid residues. The kinetic parameters of His₆-LeuRS-C were determined. The secondary structure estimated by CD spectrum and thermal stability of the insertion mutant was compared with those of His₆-LeuRS, respectively.