Obilježavanje S-adenozil-metionin sintetaze hemaglutininskom-oznakom i njezina detekcija na površini stanične stijenke kvasca

undergraduate thesis

• Title (english): HA-labeling of S-adenosyl-methionine synthetase and its detection on the surface of the yeast cell wall
• Author: Anđela Vukšić
• Mentor: Bojan Žunar (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2023-09-08, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Izlaganje proteina na staničnu stijenku kvasca inovativna je tehnika korištenja enzima u biotehnološkoj proizvodnji. Kako bi se ispitalo može li se na staničnoj stijenci kvasca Saccharomyces cerevisiae izložiti i na nju kovalentno vezati kvaščev enzim S-adenozil-metionin sintetaza (Sam1), u ovom radu konstruiran je plazmid pRSII423-ccw12sam1-3ha, koji kodira za hemaglutininskom oznakom obilježen fuzijski protein Sam1-Ccw12. Navedeni fuzijski protein, spoj enzima Sam1 i Cterminalnog dijela proteina stanične stijenke Ccw12, zatim je eksprimiran u stanicama kvasca, nakon čega je imunoblotom pokazano da on uspješno prolazi kroz sekretorni put kvasca te se nekovalentno i kovalentno, preko GPI sidra, veže na staničnu stijenku kvasca. Ovim rezultatima po prvi je puta pokazano da je enzim S-adenozil-metionin sintetazu moguće izložiti na površini stanične stijenke kvasca S. cerevisiae.

Abstract (english)

Displaying proteins on the yeast cell wall is an innovative technique for the use of enzymes in biotechnological production. To investigate the possibility of displaying and covalently attaching the yeast enzyme S-adenosylmethionine synthase (Sam1) to the cell wall of yeast Saccharomyces cerevisiae, a plasmid named pRSII423-ccw12sam1-3ha was constructed in this study. This plasmid encodes a fusion protein, Sam1-Ccw12, which is marked with a hemagglutinin tag. This fusion protein, a combination of the Sam1 enzyme and the C-terminal part of the cell wall protein Ccw12, was then expressed in yeast cells. Immunoblot analysis demonstrated that this fusion protein successfully traverses the yeast's secretory pathway and becomes non-covalently and covalently attached to the yeast cell wall via a GPI anchor. These results demonstrate, for the first time, that it is feasible to expose the enzyme S-adenosylmethionine synthase on the cell wall surface of the yeast S. cerevisiae.

• Keywords: izlaganje proteina na staničnu stijenku; hemaglutininska oznaka; S-adenozil-metionin sintetaza; stanična stijenka; kvasac Saccharomyces cerevisiae
• Keywords (english): surface display; hemagglutinin tag; S-adenosyl-methionine synthetase; cell wall; yeast Saccharomyces cerevisiae
• Language: croatian
• URN:NBN: urn:nbn:hr:159:292823
• 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: 2024-04-30 08:52:56