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Default Overproduction of the N-terminal Anticodon-binding Domain of the Non-discriminating Aspartyl-tRNA Synthetase from Helicobacter pylori for Crystallization and NMR Measurements.

Overproduction of the N-terminal Anticodon-binding Domain of the Non-discriminating Aspartyl-tRNA Synthetase from Helicobacter pylori for Crystallization and NMR Measurements.

Overproduction of the N-terminal Anticodon-binding Domain of the Non-discriminating Aspartyl-tRNA Synthetase from Helicobacter pylori for Crystallization and NMR Measurements.

Protein Expr Purif. 2013 Feb 27;

Authors: Fuengfuloy P, Chuawong P, Suebka S, Wattana-Amorn P, Williams C, Crump MP, Songsiriritthigul C

Abstract
Aminoacyl-tRNA synthetases (aaRSs) covalently attach an amino acid to its cognate tRNA isoacceptors through an ester bond. The standard set of 20 amino acids implies 20 aaRSs for each pair of amino acid/tRNA isoacceptors. However, the genomes of all archaea and some bacteria do not encode for a complete set of 20 aaRSs. For the human pathogenic bacterium Helicobacter pylori, a gene encoding asparaginyl-tRNA synthetase (AsnRS) is absent whilst an aspartyl-tRNA synthetase (AspRS) aminoacylates both tRNAAsp and tRNAAsn with aspartate. The structural and functional basis for this non-discriminatory behavior is not well understood. Here we report the over-production of the N-terminal anticodon-binding domain of H. pylori ND-AspRS using Escherichia coli BL21(DE3) host cells. Prolonged expression of this protein resulted in a toxic phenotype, limiting the expression period to just 30 minutes. Purified protein was monomeric in solution by gel filtration chromatography and stable up to 42 oC as observed in temperature-dependent dynamic light scattering measurements. Circular dichroism indicated a mixture of ?-helix and ?-sheet secondary structure at 20 oC and predominantly ?-sheet at 70 oC. Optimized crystallization conditions at pH 5.6 with PEG 4000 as a co-precipitant produced well-formed crystals and 1H NMR spectrum shows a well dispersed chemical shift envelope characteristic of a folded protein.


PMID: 23454362 [PubMed - as supplied by publisher]



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