Expression, purification, crystallization and preliminary x-ray crystallographic analysis of CnrX from Cupriavidus metallidurans CH34

Abstract

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 13.6 kDa by size exclusion chromatography, corresponding to a monomer. The tetragonal bipyramid crystals were obtained by mixing an equal volume of protein in 50 mM Tris-HCl, pH 7.5, 1% glycerol, 100 mM NaCl, I mM DTT, and the reservoir solution of 15% w/v PEG 2000, 100 mM lithium chloride at 277 K in 2-4 days using hanging drop vapor diffusion. The protein concentration was 24 mg/ml. The crystal that diffracted to 2.42 angstrom resolution belongs to space group P4(1) or P4(3) with unit cell parameters of a=b=32.14 angstrom, c= 195.31 angstrom, alpha=beta=gamma=90 degrees, with one molecule of CnrX in the asymmetric unit.