Expression, purification, crystallization and preliminary X-ray diffraction analysis of a ribokinase from the thermohalophile Halothermothrix orenii.
A ribokinase gene (rbk) from the anaerobic halothermophilic bacterium Halothermothrix orenii was cloned and overexpressed in Escherichia coli. The recombinant protein (Ho-Rbk) was purified using immobilized metal-ion affinity chromatography and crystals were obtained using the sitting-drop method. Diffraction data were collected to a resolution of 3.1 Å using synchrotron radiation. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 45.6, b = 61.1, c = 220.2, and contained two molecules per asymmetric unit. A molecular-replacement solution has been found and attempts are currently under way to build a model of the ribokinase. Efforts to improve crystal quality so that higher resolution data can be obtained are also being considered.
Microbial Gene Research and Resources Facility, School of Biomolecular and Physical Sciences, Griffith University, Brisbane, QLD 4111, Australia.
Lokesh D
LD
Kori
Andreas
A
Hofmann
Bharat K C
BK
Patel
D001419:Bacteria / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D017853:Phosphotransferases (Alcohol Group Acceptor); C022534:ribokinase