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Expression, Purification, Crystallization and Preliminary X-Ray Crystallographic Analysis of CnrX from Cupriavidus metallidurans CH34
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  • Expression, Purification, Crystallization and Preliminary X-Ray Crystallographic Analysis of CnrX from Cupriavidus metallidurans CH34
저자명
Kim. Kook-Han,Jung. Eun-Jung,Im. Ha-Na,Lelie. Daniel Van Der,Kim. Eunice Eun-Kyeong
간행물명
Journal of microbiology and biotechnology
권/호정보
2008년|18권 1호|pp.43-47 (5 pages)
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한국미생물생명공학회
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이 논문은 한국과학기술정보연구원과 논문 연계를 통해 무료로 제공되는 원문입니다.
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기타언어초록

The nickel and cobalt resistance of Cupriavidus metallidurans CH34 is mediated by the CnrCBA efflux pump encoded by the cnrYHXCBAT metal resistance determinant. The products of the three genes cnrYXH transcriptionally regulate expression of cnr. CnrY and CnrX are membrane-bound proteins, probably functioning as anti-sigma factors, whereas CnrH is a cnr-specific extracytoplasmic functions (ECF) sigma factor. The periplasmic domain of CnrX (residues 29-148) was cloned as a N-terminal His-tagged protein, expressed in Escherichia coli, and purified using affinity chromatography and gel filtration. The molecular mass was estimated to be about 13.6kDa by size exclusion chromatography, corresponding to a monomer. The tetragonal bipyramid crystals were obtained by mixing an equal volume of protein in 50mM Tris-HCl, pH 7.5, 1% glycerol, 100mM NaCl, 1mM DTT, and the reservoir solution of 15% w/v PEG 2000, 100mM lithium chloride at 277K in 2-4 days using hanging drop vapor diffusion. The protein concentration was 24mg/ml. The crystal that diffracted to $2.42{AA}$ resolution belongs to space group $P4_1;or;P4_3$ with unit cell parameters of $a=b=32.14{AA},;c=195.31{AA},;{alpha}={eta}={gamma}=90^{circ}$, with one molecule of CnrX in the asymmetric unit.