engleski

CHARACTERIZATION OF CrdA - THE COPPER HOMEOSTASIS FACTOR FROM BACTERIUM HELICOBACTER PYLORI

Helicobacter pylori is currently the only known bacterium that can survive under the highly acidic conditions of the human stomach. The colonization and survival of H. pylori requires proteins that are involved in maintaining cytoplasmic metal ion homeostasis. CrdA acts as a copper resistance determinant together with CrdB and CzcB, keeping the concentration of free copper ions in the cytoplasm below toxic levels (1). We performed structural studies on the CrdA metalloprotein. CrdA was overexpressed in BL21 E. coli cells with overnight cultures and optimal expression conditions were established. In addition, recombinant proteins (His / Strep/ GST tagged) were first purified by affinity chromatography and then by size-exclusion chromatography. High expression levels of CrdA were obtained but with 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. The affinity of copper ions towards CrdA was also investigated by the UV-VIS titration. Crystallization trials were carried out by the sitting drop vapor diffusion technique using an automated crystallization platform (Oryx 8 robot) and commercial screening kits. The obtained crystals were too small for the X-ray diffraction measurements so further optimization is needed.

Helicobacter pylori; CrdA; copper

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