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undergraduate thesis
Search for interaction partners of the IleRS C-terminal domain
Zagreb: University of Zagreb, Faculty of Science, 2023. urn:nbn:hr:217:915186
Škrobo, Petar
University of Zagreb
Faculty of Science
Department of Biology

Institutional repository: Repository of the Faculty of Science

Cite this document

Škrobo, P. (2023). Search for interaction partners of the IleRS C-terminal domain (Undergraduate thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:915186

Škrobo, Petar. "Search for interaction partners of the IleRS C-terminal domain." Undergraduate thesis, University of Zagreb, Faculty of Science, 2023. https://urn.nsk.hr/urn:nbn:hr:217:915186

Škrobo, Petar. "Search for interaction partners of the IleRS C-terminal domain." Undergraduate thesis, University of Zagreb, Faculty of Science, 2023. https://urn.nsk.hr/urn:nbn:hr:217:915186

Škrobo, P. (2023). 'Search for interaction partners of the IleRS C-terminal domain', Undergraduate thesis, University of Zagreb, Faculty of Science, accessed 19 May 2024, https://urn.nsk.hr/urn:nbn:hr:217:915186

Škrobo P. Search for interaction partners of the IleRS C-terminal domain [Undergraduate thesis]. Zagreb: University of Zagreb, Faculty of Science; 2023 [cited 2024 May 19] Available at: https://urn.nsk.hr/urn:nbn:hr:217:915186

P. Škrobo, "Search for interaction partners of the IleRS C-terminal domain", Undergraduate thesis, University of Zagreb, Faculty of Science, Zagreb, 2023. Available at: https://urn.nsk.hr/urn:nbn:hr:217:915186

Metadata
TitleSearch for interaction partners of the IleRS C-terminal domain
Title (croatian)Potraga za interakcijskim partnerima Cterminalne domene IleRS
AuthorPetar Škrobo
MentorMorana Dulić (mentor)
GranterUniversity of Zagreb
Faculty of Science
(Department of Biology)
Zagreb
Defense date and country2023-07-20, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Biology
Abstract
Isoleucyl-tRNA synthetase (IleRS) is an enzyme that binds isoleucine and its cognate tRNA molecule. The bacterium Priestia megaterium has two IleRS isoenzymes, PmIleRS1 and PmIleRS2. A prominent difference between these isoenzymes is a zinc finger domain at the Cterminus of PmIleRS1 that is not present in PmIleRS2. To determine if this domain has any protein interaction partners, PmIleRS1 was overexpressed in Escherichia coli, isolated, purified by Ni-NTA agarose affinity chromatography and concentrated with ultracentrifugation. The same was done with the PmIleRS1ΔZnF protein in which the zinc finger domain is missing. Then both proteins were used as bait in Ni-NTA pull down assays. As a negative control pull-down assays were done with leucyl-tRNA synthetase as well as without bait protein. The collected fractions were analysed by SDS-PAGE and the gels were stained with Coomassie Brilliant Blue R-250. To enhance sensitivity and potentially detect low amount of protein, the electrophoresis was repeated, only this time the gel was stained with silver. The results suggest that there is no specific interaction partner of the zinc finger domain of IleRS1 from P. megaterium.
Abstract (croatian)
Izoleucil-tRNA sintetaza (IleRS) enzim je koji veže izoleucin i pripadnu tRNA molekulu. Bakterija Priestia megaterium ima dva IleRS izoenzima, PmIleRS1 i PmIleRS2. Značajna razlika između ovih izoenzima jest domena cinkovog prsta na C-terminalnom dijelu PmIleRS1 koja nije prisutna u PmIleRS2. Kako bi se odredilo ima li ta domena neke proteinske interakcijske partnere, PmIleRS1 prekomjerno je eksprimiran u bakteriji Escherichia coli, izoliran, pročišćen Ni-NTA agaroznom afinitetnom kromatografijom i ukoncentriran ultracentrifugiranjem. Isto je učinjeno s proteinom PmIleRS1ΔZnF kojem nedostaje domena cinkovog prsta. Oba su proteina korištena kao mamac u Ni-NTA metodi pull-down. Kao negativna kontrola napravljen je pull-down s leucil-tRNA sintetazom kao mamcem i još jedan bez proteina mamca. Skupljene frakcije analizirane su metodom SDS-PAGE i gelovi su obojani s Coomassie Brilliant Blue R-250. Kako bi se povećala osjetljivost i potencijalno uočili proteini u niskim koncentracijama elektroforeza je ponovljena, a gel je obojan srebrom. Rezultati ukazuju na to da ne postoji specifični interakcijski partner domene cinkovog prsta u proteinu IleRS1 iz bakterije P. megaterium.
Keywords
Keywords (croatian)
Languageenglish
URN:NBNurn:nbn:hr:217:915186
Study programmeTitle: Molecular Biology
Study programme type: university
Study level: undergraduate
Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) molekularne biologije (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) molekularne biologije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
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Created on2023-09-12 11:21:51