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Molecular cloning, sequencing and expression in Escherichia coli cells Thermus thermophilus leucyl-tRNA synthetase

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Author(s): Yaremchuk A. D. | Kovalenko O. P. | Gudzera O. I. | Tukalo M. A.

Journal: Biopolymers and Cell
ISSN 0233-7657

Volume: 27;
Issue: 6;
Start page: 436;
Date: 2011;
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Keywords: aminoacyl-tRNA synthetases | leucyl-tRNA synthetase | Thermus thermophilus | phylogenetic tree

ABSTRACT
Aim. Cloning and sequencing of the T. thermophilus leucyl-tRNA synthetase (LeuRSTT) followed by the creation of genetically engineered construct for protein expression in E.coli cells and its purification. Methods. Searching for the LeuRSTT gene was performed by Southern blot hybridization with chromosomal DNA, where digoxigenin-labeled PCR fragments of DNA were used as probes. Results. The gene of T. thermophilus HB27 leucyl-tRNA synthetase was cloned and sequenced. The open reading frame encodes a polypeptide chain of 878 amino acid residues in length (molecular mass 101 kDa). Comparison of the amino acid sequence of T. thermophilus LeuRS with that of the enzymes from other organisms showed that LeuRSTT was a part of the group of similar enzymes of prokaryotes, formed by the proteins of protobacteriae, rickettsia and mitochondria of eukaryotes. The resulting phylogenetic tree of LeuRSs reveals dichotomous branching into two lines: prokaryotic/eukaryotic mitochondrial and arhaeal/eukaryotic cytosolic proteins. Differences between prokaryotic and arhaeal branches of the LeuRSs phylogenetic tree are primarily due to the structure of two domains of the enzyme – the editing and the C-terminal. T. thermophilus LeuRS was expressed in E. coli cells by cloning the corresponding gene into pET29b vector. Conclusions. The cloned T. thermophilus leuS gene and expressed recombinant protein will be used for structural and functional studies on LeuRSTT, including X-ray analysis of the enzyme and its mutant forms in complex with different substrates
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