Search for interaction partners of the IleRS C-terminal domain

undergraduate thesis

• Title (croatian): Potraga za interakcijskim partnerima Cterminalne domene IleRS
• Author: Petar Škrobo
• Mentor: Morana Dulić (mentor)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2023-07-20, 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: aminoacyl-tRNA synthetase; Priestia megaterium; zinc finger; pull-down assay
• Keywords (croatian): aminoacil-tRNA sintetaze; Priestia megaterium; cinkov prst; metoda pull-down
• Language: english
• URN:NBN: urn:nbn:hr:217:915186
• Study programme: Title: 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 resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-09-12 11:21:51