Ispitivanje utjecaja N-glikozilacije i endocitoze na ugradnju heterolognih proteina u staničnu stijenku kvasca Saccharomyces cerevisiae

undergraduate thesis

• Title (english): Effect of N-glycosilation and endocytosis on incorporation of heterologus proteins in the Saccharomyces cerevisiae cell wall
• Author: Nikolina Orešković
• 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: 2021-09-16, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Izlaganje proteina na staničnoj površini (˝surface display˝) predstavlja poboljšanu tehniku imobilizacije proteina te omogućuje kontinuiranu sintezu rekombinantnih proteina zahvaljujući fuziji gena koji kodira za ispitivani enzim i gena koji kodiraju za nativne proteine stanične stijenke. U ovom radu rekombinantni protein β-laktamaza (gen bla) fuzioniran je sa proteinom Pir2, odnosno sa dijelom proteina Ccw12. Pir2 veže se za stijenku svojim Nterminalnim dijelom. Dio gena CCW12, korišten u ovom radu, kodira za C-terminalnu signalnu sekvencu na koju se veže GPI-sidro, preko kojeg se protein veže u stijenku. Najveći je nedostatak ovakvog pristupa mali prinos imobiliziranih proteina u stijenku. S pretpostavkom da mutacije u N-glikozilaciji i endocitozi povećavaju učinkovitost imobilizacije ispitivan je utjecaj mutacija u N-glikozilaciji (och1 mutant i mnn mutanti) te mutacija u endocitozi (vam4 mutant i end mutanti) na efikasnost ugradnje rekombinantnog proteina u stijenku mjerenjem aktivnost β-laktamaze, što omogućuje procjenu količine imobiliziranog rekombinantnog proteina u staničnoj stijenci.

Abstract (english)

„Surface display“ system provides enhanced immobilization technique that enables countinuous synthesis of recombinant protein. This is possible because of the genetic fusion between genes encoding for native cell wall proteins and enzyme that is reasearched. In this study the enzyme β-lactamase (gene bla) and Pir2 or a fragment of Ccw12 are fuzed. Pir2 is linked to the cell wall on its N-terminal end, while Ccw12 is linked to the cell wall by GPI anchor on C- terminal region. One of the major disadvantages of this approach is low yield in protein immobilization. With the assumption that mutation in N-glycosylation and endocytosis enhances surface display system efficiency, this system was examined in mutants in Nglycosylation (och1 mutant and mnn mutants) and in mutants in endocytosis (vam4 mutant and end mutants). By measuring β-lactamase activity, it is possible to estimate the amount of recombinant protein immobilized in cell wall.

• Keywords: ˝surface display˝ sustav; N-glikozilacija; endocitoza; Pir2; Ccw12
• Keywords (english): „surface display“ system; N-glycosylation; endocytosis; Pir2; Ccw12
• Language: croatian
• URN:NBN: urn:nbn:hr:159:832036
• 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: 2021-09-29 09:15:20