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Cloning, purification and crystallization of FlgD and CrdA – Helicobacter pylori survival required proteins

The Helicobacter pylori flagellum is a complex protein consisting of about 50 proteins for proper regulation and assembly. Major sections that form the flagellum are: the filament, the hook and the basal body.1 FlgD plays an important role in H. pylori virulence because its significance to form the hook cap structure2. On the other hand, CrdA acts as a metal homeostasis factor, maintaining the copper concentration in the periplasmic space under toxic levels.3 In order to clarify the structural properties and function of this two H. pylori survival required proteins we performed cloning, purification and crystallization studies. Both proteins were overexpressed in BL21 E. coli cells with overnight cultures, and optimal expression conditions were established. Afterwards, recombinant proteins (His/GST tagged) were purified firstly by affinity chromatography and then by size-exclusion chromatography. FlgD was concentrated to 30 mg/ml and the crystallization tests were performed by sitting drop vapor diffusion technique using an automated crystallization platform (Oryx 8 robot) and commercial screening kits. Crystallization of the FlgD yielded diffraction quality crystals, and Se- Met labeled FlgD was crystallized in the same condition. In the case of CrdA we obtained high expression levels but poor solubility, so refolding methods were implemented with success. CrdA protein was characterized by circular dichroism and monodimensional NMR, that confirmed the presence of secondary structure elements. Crystallization trials carried out resulted in non-diffracting crystals. In order to improve the chance for successful crystallization the GST- fusion-CrdA protein was cloned and expressed. [1] H. Zhou et al., Proteins. (2011), 2346-2351. [2] W. T. Kuo et al., J. Mol. Biol. (2008) 381, 189- 199. [3] B. Waidner et al., J. Bacteriol. (2002) 184, 6700-6708.

Helicobacter pylori; proteins; crystallization

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