Unošenje mutacija u gen SCW4 kvasca Saccharomyces cerevisiae i ispitivanje utjecaja uvedenih mutacija na vezanje Scw4 proteina u staničnu stijenku

master's thesis

• Title (english): Introduction of mutations in the SCW4 gene of the yeast Saccharomyces cerevisiae and investigation of the influence of introduced mutations on binding of Scw4 protein in the cell wall
• Author: Dora Hrestak
• Mentor: Renata Teparić (mentor)
• Committee member: Vladimir Mrša (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2021-01-15, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Scw4 protein u stijenku kvasca Saccharomyces cerevisiae veže se kovalentnim i neokovalentnim vezama, međutim, točan mehanizam kovalentnog povezivanja i dalje je nepoznat. Polazišna je pretpostavka da je način vezanja sličan onome PIR proteina, s obzirom na jednak način ekstrakcije kovalentno vezane frakcije ovog proteina iz stijenke. Cilj ovog istraživanja bio je odrediti koja je aminokiselina odgovorna za kovalentno vezanje Scw4 u stijenku kvasca. U ciljanu regiju gena SCW4 uvedene su mutacije, a uzgoj mutanata proveden je u nepuferiranom mediju, te pri pH=4 i pH=7. Nakon uzgoja nekovalentno vezani proteini su izolirani iz stijenki pomoću vrućeg SDS-a, a kovaletno vezani pomoću 30 mM NaOH. Dobiveni proteinski ekstrakti analizirani su Western blot metodom. Iz dobivenih rezultata možemo zaključiti kako je za kovalentno vezanje Scw4 u stijenku S. cerevisiae odgovoran ostatak glutamina Q₂₄₁.

Abstract (english)

The Scw4 protein of Saccharomyces cerevisiae yeast can be bound both covalently and non-covalently to the cell wall, however, the exact mechanism of covalent binding is still unknown. The initial assumption is that the binding mechanism is similar to that of PIR proteins, given the same method of extraction of covalently bound fraction of these proteins from the yeast cell wall. The aim of this study was to determine which amino acid is responsible for covalent binding of Scw4 to the yeast wall. Mutations were introduced into the target region of the SCW4 gene, followed by the cultivation in an unbuffered medium, as well as at pH=4 and pH=7. After cultivation, non-covalently bound proteins were isolated by hot SDS, while covalently bound proteins were extracted by 30 mM NaOH. Protein extracts were analyzed by the Western blot method. The obtained results showed that glutamine Q₂₄₁ is responsible for covalent binding of Scw4 to the cell wall of S. cerevisiae.

• Keywords: japsinske proteaze; Kex2 proteaza; stanična stijenka; Saccharomyces cerevisiae; Scw4
• Keywords (english): cell wall; Kex2 protease; Saccharomyces cerevisiae; Scw4; Yapsins
• Language: croatian
• URN:NBN: urn:nbn:hr:159:593938
• Study programme: Title: Bioprocess Engineering; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva (magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-03-17 12:38:14