Naslov
Structural studies about HypB and CrdA, two Helicobacter pylori metal homeostasis key factors

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

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

Izvornik
Programme and Abstract Book / - Padova, 2012, 42-42

Skup
XI Annual Retreat - Venetian Institute of Molecular Medicine - VIMM (Advanced Biomedical Research Foundation)

Mjesto i datum
Marostica, Italija, 19.10.2012. - 20.10.2012

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
Helicobacter pylori; HypB protein; CrdA protein; structure; crystallization

Sažetak
The bacterium Helicobacter pylori colonizes the stomach of more than half of the world’s population, with the highest rates in developing countries, making it one of the most successful bacterial pathogens. Due to its pathological relevance, H. pylori has become an important target for research in the last twenty years, either from the medical or from the biological point of view. In this investigation we focused on the structural studies of HypB and CrdA, two proteins that are involved in maintaining cytoplasmic metal ion (Ni or Cu) homeostasis and that play a central role in the adaptation of H. pylori to the changing gastric environment. Furthermore, HypB is a maturation factor of [NiFe]hydrogenase and urease, two enzymes responsible for a successful colonization of the human gastric mucosa by H. pylori. In order to investigate the structural properties of these proteins, we performed cloning, expression, purification and crystallization trials. CrdA sequence was cloned both with a Strep-tag at the N terminus and with a His- tag at the C terminus of the construct, while the HypB sequence was cloned only as a Strep-tagged construct. Both proteins were expressed in BL21 E. coli cells with overnight cultures grown at 28°C. Furthermore, recombinant proteins were purified firstly by affinity chromatography and then by gel filtration. For the HypB protein an anaerobic treatment with reducing agents was performed using glove box. Afterwards, a reduced HypB was incubated with nickel. In the case of CrdAHis-tag and Strep-tagCrdA protein, we obtained high expression levels but poor solubility, so refolding methods were implemented with success. Incubation of the CrdA protein with copper followed by gel filtration was carried out. Crystallization trials were performed for HypB and CrdA proteins using an automated crystallization platform (Oryx 8 robot).

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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