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 language | English (US) |
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Pages (from-to) | 146-149 |
Number of pages | 4 |
Journal | Protein Expression and Purification |
Volume | 36 |
Issue number | 1 |
DOIs | |
State | Published - Jul 2004 |
Externally published | Yes |
Keywords
- Aquifex aeolicus
- Escherichia coli
- Leucyl-tRNA synthetase
- Overproduction
- argU
ASJC Scopus subject areas
- Biotechnology