Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of CheY3, a response regulator that directly interacts with the flagellar 'switch complex' in Vibrio cholerae

Abstract

Vibrio cholerae is the aetiological agent of the severe diarrhoeal disease cholera. This highly motile organism uses the processes of motility and chemotaxis to travel and colonize the intestinal epithelium. Chemotaxis in V. cholerae is far more complex than that in Escherichia coli or Salmonella typhimurium, with multiple paralogues of various chemotaxis genes. In contrast to the single copy of the chemotaxis response-regulator protein CheY in E. coli, V. cholerae contains four CheYs (CheY1-CheY4), of which CheY3 is primarily responsible for interacting with the flagellar motor protein FliM, which is one of the major constituents of the ;switch complex' in the flagellar motor. This interaction is the key step that controls flagellar rotation in response to environmental stimuli. CheY3 has been cloned, overexpressed and purified by Ni-NTA affinity chromatography followed by gel filtration. Crystals of CheY3 were grown in space group R3, with a calculated Matthews coefficient of 2.33 A3 Da(-1) (47% solvent content) assuming the presence of one molecule per asymmetric unit.

Figure 1

(a) Confirmation of the CheY3 clone by restriction digestion with NdeI and BamHI, showing a band at 405 bp. Lane 1, 100 bp ladder; lane 2, CheY3 clone. (b) Overexpression of His-tagged CheY3 showing uninduced and induced lanes (1 and 2, respectively) together with molecular-weight markers (lane 3; labelled in kDa) on 15% SDS–PAGE. (c) The homogeneity of the purified CheY3 was checked by 15% SDS–PAGE. CheY3 is shown in lane S; lane M contains molecular-weight markers (kDa).

Figure 2

Crystals of CheY3 grown (a) in the presence of high-salt precipitant solution consisting of 0.8 M ammonium sulfate in 0.1 M HEPES pH 7.0 and (b) in low-salt precipitant solution consisting of 5% PEG 6000 in 0.1 M Tris–HCl pH 8.0.

Figure 3

X-ray diffraction image of the CheY3 crystal grown under low-salt conditions; the crystal diffracts to a highest resolution of 1.86 Å.