title: Overproduction of the N-terminal anticodon-binding domain of the non-discriminating aspartyl-tRNA synthetase from Helicobacter pylori for crystallization and NMR measurements.
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.
authors: Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Kasetsart University, 50 Ngamwongwan Rd., Chatuchak, Bangkok 10900, Thailand.
authors: Pitchayada
authors: P
authors: Fuengfuloy
authors: Pitak
authors: P
authors: Chuawong
authors: Suwimon
authors: S
authors: Suebka
authors: Pakorn
authors: P
authors: Wattana-Amorn
authors: Christopher
authors: C
authors: Williams
authors: Matthew P
authors: MP
authors: Crump
authors: Chomphunuch
authors: C
authors: Songsiriritthigul
mesh_terms: D000595:Amino Acid Sequence; D000926:Anticodon; D001226:Aspartate-tRNA Ligase / Q000737:chemistry / Q000302:isolation & purification*; D001665:Binding Sites; D016481:Helicobacter Infections / Q000201:enzymology*; D016480:Helicobacter pylori / Q000201:enzymology*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012343:RNA, Transfer / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D000926:Anticodon; D016601:RNA-Binding Proteins; D012343:RNA, Transfer; D001226:Aspartate-tRNA Ligase