Gene cloning, expression, crystallization and preliminary x-ray analysis of Thermus thermophilus arginyl-tRNA synthetase

Atsushi Shimada, O. Nureki, N. Dohmae, K. Takio, S. Yokoyama

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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 P65, with unit-cell parameters a = b = 156.04 (7), c = 87.17 (4) Å. X-ray data to 2.8 Å 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 Å resolution using synchrotron radiation from the beamline 41XU at SPring-8 (Harima).

Original languageEnglish
Pages (from-to)272-275
Number of pages4
JournalActa Crystallographica Section D: Biological Crystallography
Volume57
Issue number2
DOIs
Publication statusPublished - Feb 22 2001
Externally publishedYes

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Arginine-tRNA Ligase
Thermus thermophilus
Crystallization
Organism Cloning
X-Rays
Selenomethionine
Gene Expression
Synchrotrons
Uranium
Ethylene Glycol
Platinum
Radiation
Escherichia coli
Temperature
Genes

All Science Journal Classification (ASJC) codes

  • Structural Biology

Cite this

Gene cloning, expression, crystallization and preliminary x-ray analysis of Thermus thermophilus arginyl-tRNA synthetase. / Shimada, Atsushi; Nureki, O.; Dohmae, N.; Takio, K.; Yokoyama, S.

In: Acta Crystallographica Section D: Biological Crystallography, Vol. 57, No. 2, 22.02.2001, p. 272-275.

Research output: Contribution to journalArticle

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