An Insertion Mutant of LeuRS with 116 Amino
Acid Residues Has Full Activity

HUANG Ying¹, LING Chen, LI Tong, TONG Geng-Lei¹, WANG En-Duo^*

( State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
the Chinese Academy of Sciences, Shanghai 200031, China;
¹ College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China )

Abstract 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 tRNAleu. 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.

Key words E.coli; leucyl-tRNA synthetase; insertion mutant; expression and purification; activity

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