Utjecaj uklanjanja Cwp2, Scw4 i/ili Pir proteina iz stanične stijenke na efikasnost izlaganja heterolognih proteina na površini kvasca

undergraduate thesis

• Title (english): The effect of removing Cwp2, Scw4 and/or Pir proteins from the cell wall on the efficiency of yeast surface display of heterologus proteins
• Author: Lucija Vukšić
• Mentor: Mateja Lozančić (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2022-09-07, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Tehnologija izlaganja proteina na površinu stanične stijenke temelji se na fuzioniranju gena od interesa s nekim od gena koji kodira za proteine stijenke te se tako nastali rekombinantni protein ugrađuje u stijenku kvasca. Na ovaj način moguće je eksprimirati čitave enzimske sustave čime se postiže njihova imobilizacija, lakše izdvajanje produkta te produljeno prisustvo biokatalizatora u odnosu na procese koji koriste pročišćene enzime. Cilj ovog rada jest ispitati utjecaj uklanjanja nativnih proteina iz stanične stijenke na efikasnost izlaganja heterolognih proteina na površinu stanice. U tu svrhu korišteni su konstrukti u kojima je gen bla fuzioniran sa nativnim genom PIR2 odnosno dijelom gena CCW12 te je rekombinantni protein zatim eksprimiran u stanicama divljeg tipa i u stanicama mutanata. Uspješnost ugradnje enzima u staničnu stijenku analizirana je mjerenjem aktivnosti βlaktamaze izložene na površinu stanice. Najveće povećanje aktivnosti u odnosu na stanice divljeg tipa postignuto je eksprimiranjem konstrukta CCW12bla u stanicama 4pirΔscw4Δ .

Abstract (english)

The yeast surface display technology is based on fusing the gene of interest with one of the genes that code for the proteins of the cell wall, and the resulting recombinant protein is incorporated in the wall. By using surface display it is posible to achieve expresion and imobilisation of enzymes, simplification of product purification, and prolongation of biocatalysts presence in the reactions. This work aims to examine the influence of removing native proteins from the cell wall on the efficiency of surface display. For this purpose, constructs were used in which the bla gene, coding for beta-lactamase, was fused with the PIR2 or fragment of CCW12 gene. The recombinant protein was expressed in wildtype and mutated cells. Efficiency of enzyme incorporation into the wall was analyzed by measuring βlactamase activity. Highest increase of activity compaired to wild type was measured in 4pirΔscw4Δ mutant when CCW12bla construct was expressed.

• Keywords: Saccharomyces cerevisiae; izlaganje proteina na staničnoj površini; Pir proteini; Scw4; Cwp2
• Keywords (english): Saccharomyces cerevisiae; yeast surface display; Pir proteins; Scw4; Cwp2
• Language: croatian
• URN:NBN: urn:nbn:hr:159:845886
• 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: 2024-04-08 09:13:41