[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 His(6)-tag at its N-terminus was constructed to facilitate the purification of His(6)-LeuRS-C through one-step affinity chromatography on Ni(2+)-NTA column. The purified His(6)-LeuRS-C had full activity as the native LeuRS with His-tag at the N-terminus (His(6)-LeuRS), although the mutant enzyme had an insertion of 116 amino acid residues. The kinetic parameters of His(6)-LeuRS-C were determined. The secondary structure estimated by CD spectrum and thermal stability of the insertion mutant was compared with those of His(6)-LeuRS, respectively.
Huang
Ying
Y
College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Ling
Chen
C
Li
Tong
T
Tong
Geng-Lei
GL
Wang
En-Duo
ED
D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D007935:Leucine-tRNA Ligase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D010446:Peptide Fragments; D011994:Recombinant Proteins; D007935:Leucine-tRNA Ligase; D007930:Leucine