Gene cloning, expression, crystallization and preliminary X-ray analysis of Thermus thermophilus arginyl-tRNA synthetase.
The gene encoding the highly thermostable arginyl-tRNA synthetase (ArgRS) from Thermus thermophilus was cloned and overexpressed in Escherichia coli under the control of the T7 promoter. The recombinant ArgRS was purified by two chromatographic steps and was crystallized by the hanging-drop vapour-diffusion method using PEG 8000 and ethylene glycol as precipitants. The crystals belong to the hexagonal space group P6(5), with unit-cell parameters a = b = 156.04 (7), c = 87.17 (4) A. X-ray data to 2.8 A resolution were collected at room temperature from a native crystal using an in-house X-ray source. Uranium, platinum and selenomethionine derivatives were found to be useful for phasing by the multiple isomorphous replacement method with anomalous scattering. The flash-frozen crystals diffracted beyond 2.3 A resolution using synchrotron radiation from the beamline 41XU at SPring-8 (Harima).
Shimada
A
A
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Nureki
O
O
Dohmae
N
N
Takio
K
K
Yokoyama
S
S
D000595:Amino Acid Sequence; D001126:Arginine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli; D019855:Ethylene Glycol; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D020959:Polyethylene; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D007202:Indicators and Reagents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D020959:Polyethylene; D001126:Arginine-tRNA Ligase; D019855:Ethylene Glycol