Ispitivanje načina vezanja mutiranog proteina Scw4 u staničnu stijenku kvasca Saccharomyces cerevisiae

undergraduate thesis

• Title (english): Examination of the way of binding of the mutated Scw4 protein into the cell wall of yeast Saccharomyces cerevisiae
• Author: Ana Matičević
• Mentor: Renata Teparić (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2020-07-10, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Protein Scw4 u staničnu stijenku kvasca Saccharomyces cerevisiae može se vezati nekovalentno i kovalentno, no i dalje nije poznato koji dio proteina je odgovoran za kovalentno povezivanje. U prethodnom istraživanju uvedena je mutacija u jednu od regija koja je slična Pir repetitivnim regijama za koje se pretpostavlja da bi mogle biti odgovorne za kovalentno povezivanje. Također je utvrđeno kako se nativni Scw4 u staničnoj stijenci pojavljuje u tri forme različite veličine – neprocesiranoj, procesiranoj sa Kex2 proteazom te procesiranoj sa japsinskim proteazama. U ovom radu ispitivan je način vezanja mutiranog proteina Scw4 u staničnu stijenku kvasca Saccharomyces cerevisiae pri uzgoju na pH 4 i pH 7. Prethodno izraĎen konstrukt sa mutacijom umnožen je u bakteriji Escherichia coli, izoliran te su njime transformirane kvaščeve stanice. Kvasci su uzgajani u selektivnoj podlozi za uzgoj u uvjetima indukcije GAL promotora. Nakon uzgoja provedena je izolacija i analiza načina vezanja proteina stanične stijenke metodama SDS elektroforeze te metodom western blota. Na osnovu dobivenih rezultata može se zaključiti da je ispitivana regija u sekvenci Scw4 proteina bitna za njegovo kovalentno vezanje u staničnu stijenku.

Abstract (english)

Protein Scw4 can be bound into the cell wall of yeast Saccharomyces cerevisiae non-covalently and covalently. However, the part of the protein responsible for the covalent bond remains unknown. According to previous research, a mutation has been introduced into one of the regions similar to Pir repetitive regions which are supposed to be responsible for the covalent bonding. It has also been established that the Scw4 protein can appear in the cell wall in three different sized forms – unprocessed, processed with Kex2 protease, and processed with yapsin proteases. In this particular research, the way of binding mutated Scw4 protein grown on pH 4 and pH 7 has been examined. A previously made mutated construct has been replicated in Escherichia coli, isolated, and used to transform yeast cells which have been grown on selective media under the induction of the promotor. Cell wall proteins have been isolated and analysed by SDS PAGE and by western blotting. According to the results obtained, it can be concluded that the examined region in the Scw4 protein sequence is important for its covalent binding to the cell wall.

• Keywords: Saccharomyces cerevisiae; Scw4; stanična stijenka
• Keywords (english): cell wall; Saccharomyces cerevisiae; Scw4
• Language: croatian
• URN:NBN: urn:nbn:hr:159:050974
• Study programme: Title: Biotechnology; Study programme type: university; Study level: undergraduate; Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2020-07-22 09:02:20