High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Chen Ling, Yong Gang Zheng, En Duo Wang

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5kDa, and named αβ-LeuRS. The gene encoding α subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of α subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNAArg(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding β subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce αβ-LeuRS with a His6 tag at the N-terminus of α subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7mg purified αβ-LeuRS was obtained from 250ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.

Original languageEnglish (US)
Pages (from-to)146-149
Number of pages4
JournalProtein Expression and Purification
Volume36
Issue number1
DOIs
StatePublished - Jul 1 2004
Externally publishedYes

Keywords

  • Aquifex aeolicus
  • Escherichia coli
  • Leucyl-tRNA synthetase
  • Overproduction
  • argU

ASJC Scopus subject areas

  • Biotechnology

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