title: High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus.
abstract: Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5 kDa, and named alphabeta-LeuRS. The gene encoding alpha subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of alpha subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNA(Arg)(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 beta subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce alphabeta-LeuRS with a His6 tag at the N-terminus of alpha subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7 mg purified alphabeta-LeuRS was obtained from 250 ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, 200031, PR China.
authors.forename: Chen
authors.initials: C
authors.lastname: Ling
authors.forename: Yong-Gang
authors.initials: YG
authors.lastname: Zheng
authors.forename: En-Duo
authors.initials: ED
authors.lastname: Wang
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D007935:Leucine-tRNA Ligase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D046249:Transfer RNA Aminoacylation
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006639:Histidine; D007935:Leucine-tRNA Ligase