Priprava rekombinantnega proteina RuBisCO aktivaza navadnega repnjakovca

Auer, Špela (author), Taler-Verčič, Ajda (mentor)

O vlogi joda v rastlinah je znano zelo malo. Ta v njih prevladuje v obliki jodida (I-), nekaj pa ga je vezanega v organske molekule. Dodajanje joda pri gojenju poveča biomaso rastline in pospeši čas cvetenja. Nekateri proteini v navadnem repnjakovcu (Arabidopsis thaliana) imajo jodirane tirozinske aminokislinske ostanke. Mednje spada tudi RuBisCO aktivaza, ki je kloroplastni encim in ga uvrščamo v skupino katalitičnih šaperonov. Je ATPaza, ki spremeni konformacijo encima RuBisCO tako, da ta preide iz zaprte v odprto konformacijo in pri tem se iz aktivnega mesta sprosti inhibitorska molekula. Namen našega dela je bilo pripraviti in izolirati topen protein RuBisCO aktivaza navadnega repnjakovca v bakterijskem ekspresijskem sistemu. Želeli smo izraziti celotno in skrajšano različico proteina. Skrajšana različica je imela odstranjenih 35 aminokislinskih ostankov iz C-konca. Najprej smo pripravili fagmid s skrajšanim zapisom za RuBisCO aktivazo, potem smo obe različici proteina izrazili in izolirali ter očistili z nikelj afinitetno kromatografijo. Ugotovili smo, da protein RuBisCO aktivaza lahko pripravimo z uporabo bakterijskega ekspresijskega sistema. Vendar nam je uspelo v zadostnih količinah za nadaljnje raziskave izraziti le celoten protein, pri skrajšani različici je bilo proteina zelo malo oziroma ga ni bilo. V prihodnje bi morali optimizirati celoten postopek izražanja skrajšane RuBisCO aktivaze.

• Language: Slovenian
• Keywords: RuBisCO aktivaza, jodiranje, molekulsko kloniranje, nikelj afinitetna kromatografija
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2025
• PID: 20.500.12556/RUL-166646
• COBISS.SI-ID: 225431555
• Publication date in RUL: 20.01.2025

AUER, Špela, 2025, Priprava rekombinantnega proteina RuBisCO aktivaza navadnega repnjakovca [online]. Bachelor’s thesis. [Accessed 14 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=166646

Secondary language

• Language: English
• Title: Preparation of the recombinant protein RuBisCO activase from the plant Arabidopsis thaliana

Abstract

Very little is known about the role of iodine in plants. Iodine is predominantly present in plants in the form of iodide (I-), but some is bound to organic molecules. The addition of iodine in cultivation increases the biomass of the plant and accelerates the flowering time. Some of the proteins in Arabidopsis thaliana have iodinated tyrosine residues. These include RuBisCO activase, a chloroplast enzyme that belongs to the group of catalytic chaperones. It is an ATPase that changes the conformation of the RuBisCO enzyme from a closed to an open conformation and thus enables the release of an inhibitory molecule from the active site. The aim of our work was to express and isolate a soluble protein of RuBisCO activase (from Arabidopsis thaliana) in a bacterial expression system. We wanted to express the protein in two lengths: a standard version and a truncated version. In the truncated version, 35 amino acid residues were removed from the C-terminus. First, we prepared a phagemid with a truncated sequence for RuBisCO activase. Then both versions of the protein were expressed, isolated and purified by nickel affinity chromatography. We found that the RuBisCO activase protein can be produced using a bacterial expression system. However, we were only able to express RuBisCO activase in sufficient quantities for further studies, while the expression of the truncated version was not effective. In the future, we should optimise the whole process of expressing the truncated version of RuBisCO activase.

• Keywords: RuBisCO activase, iodination, molecular cloning, nickel affinity chromatography