Pojačana ekspresija, pročišćavanje i kristalizacija proteina CrdA te optimizacija kristalizacije proteina HP1026 iz bakterije Helicobacter pylori

master's thesis

• Title (english): Overexpression, purification and crystallization of the CrdA protein and optimization of crystallization of the HP1026 protein from the bacterium Helicobacter pylori
• Author: Vigor Matković
• Mentor: Dubravka Matković-Čalogović (mentor)
• Committee member: Dubravka Matković-Čalogović (predsjednik povjerenstva)
• Committee member: Sanda Rončević (član povjerenstva)
• Committee member: Morana Dulić (član povjerenstva)
• Committee member: Biserka Prugovečki (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2018-07-10, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

Bakterija Helicobacter pylori je Gram-negativna spiralna bakterija koja u čovjeku kolonizira stijenku želuca te dugoročnom infekcijom s bakterijom (pogotovo soj P12) dolazi do razvoja mnogih oboljenja gornjeg gastrointestinalnog trakta. Izvršena je pojačana ekspresija proteina HP1026 (helikaze odgovorne za rekombinantne aktivnosti) iz soja P12 H. pylori, pročišćavanje te kristalizacija metodom difuzije para tehnikom sjedeće kapi (engl. "sitting drop"). Na XRD1 stanici sinkrotrona „Elettra Sincrotrone Trieste“ istražena je kvaliteta 20 kristala s obzirom na difrakciju rentgenskih zraka na monokristalu. Najbolji difrakcijski podaci sakupljeni su do razlučenja od 4 Å u heksagonskom kristalnom sustavu i s parametrima jedinične ćelije od a = 116,22(2) Å, c = 238,38(2) Å. Također je predložena vrlo jednostavna i uspješna metoda kloniranja gena crdA, indukcije pojačane ekspresije, pročišćavanja te kristalizacije proteina CrdA iz H. pylori soja P12 prethodnom pripravom fuzijskog SUMO proteina. U konačnici je dobiven čist i koncentriran protein CrdA u otopini, koji se može koristiti dalje u kristalizacijskim eksperimentima.

Abstract (english)

Helicobacter pylori is a Gram-negative spiral bacterium that colonizes the stomach cells, manages long-term bacterial infections (especially strain P12) and develops many diseases of the upper gastrointestinal tract. Overexpression of HP1026 (helicase responsible for recombinant activity) protein from P12 H. pylori strain, purification and crystallization using the "sitting drop" technique was performed. At the XRD1 beamline of the synchrotron "Elettra Sincrotrone Trieste", the quality of 20 crystals was investigated by X-ray diffraction on single crystals. The best diffraction data obtained had the resolution of 4 Å, in the hexagonal crystal system with unit cell parameters of a = 116.22(2), c = 238.38(2) Å. A very simple and successful method of cloning the crdA gene by creating the SUMO fusion protein was done, followed by protein overexpression, purification and crystallization of the CrdA protein from the H. pylori strain P12. The obtained result was a pure and concentrated solution of the CrdA protein which can be further used in crystallization experiments.Helicobacter pylori is a Gram-negative spiral bacterium that colonizes the stomach cells, manages long-term bacterial infections (especially strain P12) and develops many diseases of the upper gastrointestinal tract. Overexpression of HP1026 (helicase responsible for recombinant activity) protein from P12 H. pylori strain, purification and crystallization using the "sitting drop" technique was performed. At the XRD1 beamline of the synchrotron "Elettra Sincrotrone Trieste", the quality of 20 crystals was investigated by X-ray diffraction on single crystals. The best diffraction data obtained had the resolution of 4 Å, in the hexagonal crystal system with unit cell parameters of a = 116.22(2), c = 238.38(2) Å. A very simple and successful method of cloning the crdA gene by creating the SUMO fusion protein was done, followed by protein overexpression, purification and crystallization of the CrdA protein from the H. pylori strain P12. The obtained result was a pure and concentrated solution of the CrdA protein which can be further used in crystallization experiments.

• Keywords: CrdA; HP1026; kloniranje; kristalizacija; pročišćavanje; SUMO
• Keywords (english): CrdA; cloning; crystallization; HP1026; purification; SUMO
• Language: croatian
• URN:NBN: urn:nbn:hr:217:729721
• Study programme: Title: Graduate university studies in chemistry; specializations in: Research programme; Course: Research programme; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-07-12 15:50:17