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

Ying Huang, Chen Ling, Tong Li, Geng Lei Tong, En Duo Wang

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)225-229
Number of pages5
JournalActa Biochimica et Biophysica Sinica
Volume35
Issue number3
StatePublished - May 19 2003
Externally publishedYes

Keywords

  • Activity
  • E. coli
  • Expression and purification
  • Insertion mutant
  • Leucyl-tRNA synthetase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry

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