Overproduction of the N-terminal anticodon-binding domain of the non-discriminating aspartyl-tRNA synthetase from Helicobacter pylori for crystallization and NMR measurements

Pitchayada Fuengfuloy, Pitak Chuawong, Suwimon Suebka, Pakorn Wattana-Amorn, Christopher Williams, Matthew P Crump, Chomphunuch Songsiriritthigul, Matthew P Crump

Research output: Contribution to journalArticle (Academic Journal)

3 Citations (Scopus)

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 tRNA(Asp) and tRNA(Asn) 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 30min. Purified protein was monomeric in solution by gel filtration chromatography and stable up to 42°C as observed in temperature-dependent dynamic light scattering measurements. Circular dichroism indicated a mixture of α-helix and β-sheet secondary structure at 20°C and predominantly β-sheet at 70°C. Optimized crystallization conditions at pH 5.6 with PEG 4000 as a co-precipitant produced well-formed crystals and (1)H NMR spectrum showed a well dispersed chemical shift envelope characteristic of a folded protein.
Original languageEnglish
Pages (from-to)25-32
Number of pages8
JournalProtein Expression and Purification
Volume89
Issue number1
DOIs
Publication statusPublished - May 2013

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Copyright © 2013 Elsevier Inc. All rights reserved.

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