Naslov
Purine nucleoside phosphorylase from bacterium Helicobacter pylori strain 26695: Cloning, Expression, Purification, Characterisation and Crystallisation

Autori
Gucunski, Karolina ; Leščić Ašler, Ivana ; Luić, Marija

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

Izvornik
29th European Crystallographic Meeting Book of Abstracts / - Zagreb, 2015

Skup
29th European Crystallographic Meeting

Mjesto i datum
Rovinj, Hrvatska, 23.08.2015. - 28.08.2015

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Helicobacter pylori; purine nucleoside phosphorylase; biochemical characterisation; enzyme kinetics; crystallisation

Sažetak
Purine nucleoside phosphorylase (PNP) is the key enzyme in the purine salvage pathway. It catalyses the reversible phosphorolytic cleavage of the glycosidic bond of ribo- and deoxyribonucleosides, in the presence of inorganic orthophosphate as a second substrate to generate the purine base and ribose(deoxyribose)-1-phosphate. Helicobacter pylori is a Gram-negative, microaerophilic bacterium, human pathogen involved in development of many diseases as gastric ulcers and stomach cancer, and therefore known for its ability to colonize human stomach (Makola et al., 2007). Study of the H. pylori, due to the ever growing infection rate and increase of H. pylori antibiotic resistance, is centred on understanding pathogenesis and finding a way to attack and eradicate H. pylori. H. pylori PNP represents potential drug target as this bacterium cannot synthesize purine rings through de novo pathway and has to rely on purine production through purine salvage pathway. It belongs to the class of bacterial high-molecular-mass homohexamers with specificity for both 6-oxo- and/or 6- aminopurines. Purine nucleoside phosphorylase gene deoD was isolated from genomic DNA of Helicobacter pylori (strain 26695) and amplified using Phusion High-Fidelity PCR kit with the set of specific DNA primers for both 5' and 3' ends of the gene. Resulting plasmid pET21b-HP26695deoD, with ampicillin resistance and without purification tag, was transformed into E. coli strain BL21-CodonPlus(DE3)RIL. Induction conditions for PNP expression in E. coli were optimised and evaluated by SDS-PAGE electrophoresis of bacterial cultrate filtrate. Purification of overexpressed PNP from the bacterial culture filtrate was performed by anion exchange chromatography on Q-Sepharose FF column. Next step, which gave single protein band on SDS-PAGE was affinity chromatography, performed on Sepharose-FormycinA column. Biochemical characterisation involves kinetic studies, as well as temperature and pH effects on stability and activity of PNP. Crystallisation experiments with purified purine nucleoside phosphorylase from H. pylori are under way.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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