Priprava in izolacija rekombinantnega proteina beta karboanhidraze rastline Arabidopsis thaliana

Kartal, Ena (author), Taler-Verčič, Ajda (mentor)

O vlogi joda pri rastlinah še ni veliko znanega. V pravih količinah vpliva na povečanje biomase in hitrejše cvetenje, vendar je lahko v prevelikih količinah za rastline tudi toksičen. Jodirane proteine najdemo v različnih delih rastline, ni pa znano, kako vezava joda na proteine vpliva na njihovo funkcijo. Po pregledu literature smo izbrali encim karboanhidrazo, ki ga najdemo tudi v jodirani obliki. Namen našega dela je bila priprava zadostne količine rekombinantnega proteina, da bi lahko vzpostavili sistem testiranja encimske aktivnosti, pri katerem bi primerjali aktivnost med tako imenovanim nativnim in jodiranim proteinom. Karboanhidraza (angl. Carbonic anhydrase) je metaloencim s cinkovim ionom v aktivnem mestu, ki katalizira medsebojno pretvarjanje CO2 in HCO3- v procesu fotosinteze. Predvsem ga lahko najdemo v listih rastlin. Pri rastlinah so prisotne tri različne družine karboanhidraz: α, β in γ. Najbolj raziskani so encimi, ki spadajo v družino β. Prisotni so v listih v dveh izooblikah: βCA1 in βCA2. Aktivno obliko predstavlja dimer, pri katerem je v aktivnem mestu cinkov ion koordiniran z dvema ostankoma cisteina, enim ostankom histidina in molekulo vode. Ti encimi imajo pomembno vlogo tudi pri rasti rastlin, sintezi aminokislin in lipidov, hkrati pa zavirajo kratkotrajne spremembe pH v stromi kloroplastov, ki jih provzročijo spremembe svetlobe ter tako delujejo pri odzivu na stres. Zapis za izbrani protein smo vstavili v vektor pET-32b(+) tako, da smo mu na 5'-konec dodali kodirajočo regijo za heksahistidinsko oznako in prepoznavno mesto za proteazo TEV. Nato smo protein pripravili v bakterijskem ekspresijskem sistemu in ga z uporabo Ni-kelatne kromatografije (IMAC) delno očistili. Ugotovili smo, da je cDNA-knjižnica iz listov rastline A. thaliana ustrezna matrica za pripravo rekombinantne karboanhidraze, ter da lahko protein v bakterijah pripravimo v zadostnih količinah za nadaljne študije. Postopki so bili uspešni le v primeru βCA1, saj nam s PCR ni uspelo pripraviti zapisa za βCA2.

• Language: Slovenian
• Keywords: beta karboanhidraza (βCA), jod, molekulsko kloniranje, bakterijski ekspresijski sistem, IMAC
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-149341
• COBISS.SI-ID: 168353283
• Publication date in RUL: 06.09.2023

Secondary language

• Language: English
• Title: Preparation and isolation of recombinant beta carbonic anhydrase protein of the plant Arabidopsis thaliana

Abstract

Not much is known about the role of iodine in plants. In the right amounts it has the effect of increasing biomass and faster flowering, but in excessive amounts it can also be toxic. Iodinated proteins are found in various parts of the plant, but it is not known how the binding of iodine to proteins affects their function. After reviewing the literature, we decided to focus on the enzyme Carbonic Anhydrase, which is also found in iodinated form. The aim of our work was to produce a sufficient amount of recombinant protein to set up a test system for enzyme activity in which the activity can be compared between the so-called normal and the iodinated protein. Carbonic anhydrase is a metalloenzyme with a zinc ion in the active site that catalyses the conversion of CO2 and HCO3- in the process of photosynthesis. It is mainly found in the leaves of plants. Three different families of carbonic anhydrase are found in plants: α, β and γ. The most studied are the enzymes of the β-family. They are present in leaves in two isoforms: βCA1 and βCA2. The active form is represented by a dimer in which the zinc ion is coordinated with two cysteine residues, a histidine residue and a water molecule in the active site. These enzymes also play an important role in plant growth, in the synthesis of amino acids and lipids, and at the same time inhibit short-term pH changes in the stroma of chloroplasts caused by light changes, and thus participate in the stress response. The transcript for the selected protein was inserted into the pET-32(+) vector with the coding region for the hexahistidine tag and the TEV protease recognition site added to the 5' end. The protein was then produced in a bacterial expression system and partially purified by Ni2+ affinity chromatography (IMAC). We have shown that the cDNA library from the leaves of the plant is a suitable starting material for the production of recombinant carbonic anhydrase and that the protein can be produced in bacteria in sufficient quantities for further studies. The procedures were only successful for βCA1, as we were unable to PCR-amplify βCA2 coding region.

• Keywords: Beta carbonic anhydrase (βCA), iodine, molecular cloning, bacterial expression system, IMAC