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) Angstrom. X-ray data to 2.8 Angstrom resolution were collected at room temperature from a native crystal using an inhouse 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 Angstrom resolution using synchrotron radiation from the beamline 41XU at SPring-8 (Harima).