Optimizacija sustava za izlaganje heterolognih proteina na površini stanica kvasca putem proteina Pir2

master's thesis

• Title (english): Optimisation of yeast surface display using protein Pir2
• Author: Ana Matičević
• Mentor: Renata Teparić (mentor)
• Committee member: Vladimir Mrša (predsjednik povjerenstva)
• Committee member: Irena Landeka Jurčević (član povjerenstva)
• Committee member: Bojan Žunar (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: 2022-07-11, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Metoda izlaganja heterolognih proteina na površini stanice kvasca Saccharomyces cerevisiae alternativa je klasičnim metodama imobilizacije proteina, a temelji se na fuziji gena koji kodira za protein od interesa sa genom koji kodira za neki od proteina stanične stijenke. Optimizacija sustava za izlaganje proteina na površini stanice kvasca u ovome je radu provedena uvođenjem promjena u sekvencu proteina Pir2 te promjenom pozicije proteina od interesa unutar sekvence proteina Pir2. U promatranom sustavu mjerenjem aktivnosti ˝reporter˝ enzima β-laktamaze te metodom imunoblota proteina stanične stijenke procijenjena je količina rekombinantnog proteina Pir2bla u staničnoj stijenki. Navedenim metodama promatran je i utjecaj mutacija u endocitozi, korištenjem mutanata end3 i end6, na efikasnost izlaganja rekombinantnog proteina na površini stanice u svrhu daljnje optimizacije sustava za imobilizaciju.

Abstract (english)

Surface display of enzymes at the cell wall of yeast Saccharomyces cerevisiae presents an alternative to chemical immobilization methods. Methods for yeast surface display are based on the fusion of a gene encoding a protein of interest with a gene encoding one of the cell wall proteins. Optimization of the protein surface display system in this paper was performed by introducing changes in the Pir2 protein sequence and changing the position of the reporter protein within the Pir2 protein sequence. In the observed system, the amount of recombinant Pir2bla protein in the cell wall was estimated by measuring the activity of the reporter enzyme β-lactamase and the immunoblot of yeast cell wall proteins. Before mentioned methods were also used to investigate the impact of mutations in endocytosis, by using mutants end3 and end6, on efficiency of surface display system in order to optimize immobilization method.

• Keywords: Pir2; stanična stijenka kvasca; izlaganje proteina na površini stanice; rekombinantni proteini; endocitoza
• Keywords (english): Pir2; yeast cell wall; yeast surface display; recombinant proteins; endocytosis
• Language: croatian
• URN:NBN: urn:nbn:hr:159:551068
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-01-19 12:23:05