Konstrukcija i mjerenje aktivnosti rekombinantnog proteina Pir2bla_CJ izloženog na površini stanične stijenke

undergraduate thesis

• Title (english): Construction and activity determination of recombinant protein Pir2bla_CJ displayed on the yeast cell surface
• Author: Dominik Matijević
• 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: 2022-06-29, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Jedna od alternativa klasičnim, unutarstaničnim, bioprocesima su izvanstanični bioprocesi temeljeni na sojevima kvasca koji izlažu biotehnološki zanimljive enzime na svojoj staničnoj površini. Takvo vezivanje proteina može se, na primjer, postići fuzijom enzima od interesa s proteinom Pir2. Međutim, nije poznato gdje je unutar proteina Pir2 najbolje umetnuti enzim, a da se što bolje sačuva i sposobnost vezanja proteina Pir2 i aktivnost izloženog enzima. U ovom radu konstruiran je plazmid pRSII-pir2bla_CJ koji kodira za fuzijski protein Pir2bla_CJ, spoj proteina Pir2 i enzima β-laktamaze. Nadalje, izmjerena je aktivnost β-laktamaze izložene na staničnoj stijenci kvasca Saccharomyces cerevisiae koji eksprimira ovaj rekombinantni protein i uspoređena s aktivnosti četiri prethodno konstruirane inačice proteina Pir2bla. Iako protein Pir2bla_CJ kombinira odlike dvije prethodno razvijene vrlo efikasne inačice proteina Pir2bla, njegova ekspresija rezultira zanemarivom aktivnošću β-laktamaze, sugerirajući da pravila koja diktiraju efikasno vezanje proteina na staničnu stijenku, uz očuvanje biotehnološki zanimljive enzimske aktivnosti, nisu aditivna.

Abstract (english)

Some classical, intracellular bioprocesses could be replaced with extracellular ones, i.e. bioprocesses based on yeast strains displaying biotechnologically interesting enzymes on their cell surface. We can bind such enzymes to the cell wall, for example, by fusing them with Pir2 proteins. However, it remains unclear where in the Pir2 protein we should insert the enzyme to preserve both the enzymatic activity and the Pir2 binding ability. In this work, we constructed a plasmid encoding protein Pir2bla_CJ, a fusion of the protein Pir2 and the enzyme β-lactamase. Furthermore, we measured the β-lactamase activity of Pir2bla_CJ on the Saccharomyces cerevisiae cell surface and compared it to four previously constructed variations of Pir2bla protein. Although Pir2bla_CJ combined characteristics of two high-performing Pir2bla variations, its β-lactamase activity was negligible, suggesting that the rules allowing for the robust binding of proteins on the cell wall, and preservation of biotechnologically relevant enzymatic activity, are not additive.

• Keywords: protein Pir2; β-laktamaza; izlaganje heterolognih proteina; stanična stijenka; kvasac Saccharomyces cerevisiae
• Keywords (english): Pir2 protein; β-lactamase; yeast surface display; cell wall; yeast Saccharomyces cerevisiae
• Language: croatian
• URN:NBN: urn:nbn:hr:159:194395
• 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: 2022-10-05 12:35:25