Naslov
Structural Characterization of the Metal Homeostasis Factors from Helicobacter Pylori

Autori
Pulić, Ivana ; Cendron, Laura ; Matković-Čalogović, Dubravka ; Zanotti, Giuseppe

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of Abstracts/ XXIII. Hrvatski skup kemičara i kemijskih inženjera / Hadžiev, Andrea ; Blažeković, Zdenko - Zagreb : Hrvatsko društvo kemijskih inženjera i tehnologa (HDKI) ; Hrvatsko kemijsko drustvo, 2013, 41-41

ISBN
978-953-6894-50-5

Skup
XXIII. Hrvatski skup kemičara i kemijskih inženjera

Mjesto i datum
Zagreb, Hrvatska, 21.04.2013. - 24.04.2013

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Domaća recenzija

Ključne riječi
Helicobacter pylori; metalloprotein; cloning; expression; purification; crystallization

Sažetak
H. pylori is currently the only known bacteria that can survive under the highly acidic conditions of the human stomach. The colonization and survival of H. pylori require proteins that are involved in maintaining cytoplasmic metal ion (Ni or Cu) homeostasis and that has a central role in the adaptation of H. pylori to the changing gastric environment. We performed structural studies of two metalloproteins HypB and CrdA. HypB is a maturation factor of [NiFe]hydrogenase and urease, two enzymes responsible for successful colonization of the human gastric mucosa by H. pylori. On the other hand, CrdA acts as a copper resistance determinant, keeping the concentration of free copper ions in the cytoplasm below toxic levels. In order to investigate the structural properties of HypB and CrdA, we performed cloning, expression, purification and crystallization trials. Both proteins were overexpressed in BL21 E. coli cells with overnight cultures and optimal expression conditions were established. In addition, recombinant proteins were purified firstly by affinity chromatography and then by size-exclusion chromatography. HypB protein was characterized by circular dichroism and dynamic light scattering, which showed good secondary structure profile but high polydispersity. 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. For both proteins, crystallization trials were carried out by the sitting drop vapor diffusion technique using an automated crystallization platform (Oryx 8 robot) and commercial screening kits. In the test with CrdA we obtained tiny crystals but unfortunately they diffracted poorly.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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