Pojačana ekspresija te pročišćavanje proteina CrdB i određivanje uvjeta kristalizacije proteina HP1026 bakterije Helicobacter pylori

master's thesis

• Title (english): Overexpression and purification of CrdB protein and determination of crystallization conditions for HP1026 protein from bacterium Helicobacter pylori
• Author: Andrea Švorinić
• Mentor: Dubravka Matković-Čalogović (mentor)
• Committee member: Dubravka Matković-Čalogović (predsjednik povjerenstva)
• Committee member: Nataša Bauer (član povjerenstva)
• Committee member: Sunčica Bosak (član povjerenstva)
• Committee member: Kristian Vlahoviček (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2017-09-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Gram-negativna patogena bakterija Helicobacter pylori kolonizira stijenke želuca preko polovine ljudske populacije. Dugoročna infekcija gotovo uvijek dovodi do razvoja kroničnog gastritisa, a u pojedinim slučajevima i do težih oboljenja poput adenokarcinoma želuca te limfoma mukoznog tkiva. U sklopu ovog rada istražena su dva proteina važna za preživljavanje i infektivni potencijal bakterije H. pylori. Protein CrdB identificiran je kao potencijalni sudionik u održavanju homeostaze iona bakra.... More Pretpostavlja se da se radi o proteinu vanjske membrane koji omogućuje izbacivanje iona bakra iz stanice, međutim struktura i funkcija ovog proteina još uvijek su neistražene. U ovom je diplomskom radu protein CrdB uspješno proizveden u obliku fuzijskog proteina SUMO-CrdB primjenom tehnologije rekombinantne DNA i prekomjernom ekspresijom u bakteriji Escherichia coli. Osmišljena je strategija pročišćavanja fuzijskog proteina SUMO-CrdB, te analizirana njegova sekundarna struktura. Pokazano je da među motivima sekundarne strukture prevladavaju β-ploče i β-okreti. Protein HP1026 je ATPaza čiji se gen nalazi pod kontrolom jednog od triju promotora toplinskog šoka bakterije H. pylori. U ovom su radu istraženi uvjeti kristalizacije proteina HP1026. Ispitivanjem različitih otopina precipitanata pronađeni su uvjeti u kojima nastaju mikrokristali proteina HP1026, stabilni kroz dulje vrijeme. Less

Abstract (english)

The bacterial pathogen Helicobacter pylori colonizes human gastric mucosa of over half of the world's population. Chronic H. pylori infection is associated with development of severe gastrointestinal diseases such as chronic gastritis, invasive adenocarcinoma and lymphoma of mucosa-associated lymphoid tissue. This thesis involves investigation of two proteins important for survival of H. pylori within the hostile environment of the human gastrointestinal tract. CrdB protein is a putative... More outer membrane efflux protein, potentially involved in maintaining copper ion homeostasis. Within this study, CrdB protein was produced as a fusion protein SUMO-CrdB by using recombinant DNA technology and overexpressed in the host bacterium Escherichia coli. A successful protein purification protocol was established. Circular dichroism was used for secondary structure determination. The analysis of circular dichroism data showed that the fusion protein SUMO-CrdB predominantly consists of β-sheets and β-turns. HP1026 is an ATPase with an interesting gene location under the control of one of the heat shock promoters. The second aim of this study was to determine crystallization conditions for HP1026 protein. Stable microcrystals were obtained after scanning a range of conditions. Less

• Keywords: Helicobacter pylori; pročišćavanje proteina; kristalizacija proteina; CrdB; HP1026
• Keywords (english): Helicobacter pylori; protein purification; protein crystallization; CrdB; HP1026
• Language: croatian
• URN:NBN: urn:nbn:hr:217:106313
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-10-20 11:51:52