diplomsko delo
Peter Škrinjar (Avtor), Brigita Lenarčič (Mentor)

Povzetek

FHL2 je adapterski protein, ki je sestavljen iz štirih in pol domen LIM, preko katerih tvori interakcije z ostalimi proteini. Med posameznimi domenami LIM se nahajajo intrinzično neurejene regije, ki dajejo proteinu fleksibilnost in mu s tem omogočajo vezavo s široko paleto različnih proteinov. Zaradi tega je FHL2 del številnih signalnih poti, ki so povezane z različnimi celičnimi procesi. Vidno vlogo ima tudi pri celičnem signaliziranju preko molekule EpCAM, ki se povišano izraža v rakavih celicah. Kot adapterski protein je FHL2 ključen pri tvorbi kompleksa EpIC-FHL2-β-katenin, ki nastane v citoplazmi in se nato prenese v jedro, kjer z vezavo na transkripcijski faktor Lef-1 sproži prepisovanje onkogenov, ki povzročijo proliferacijo celic. Mehanizem tvorbe kompleksa EpIC-FHL2-β-katenin ni razjasnjen, prav tako ni znana niti njegova struktura. Cilj našega dela je bil optimizirati izražanje rekombinantnega proteina FHL2 in ga pripraviti v zadostnih količinah, da bi ga lahko uporabili za strukturno in biokemijsko karakterizacijo kompleksa EpIC-FHL2-β-katenin. FHL2 se po induciranem izražanju v bakterijskih celicah nahaja v netopni frakciji, zato ga je potrebno izolirati iz inkluzijskih telesc. V sklopu našega raziskovalnega dela smo izvedli tri različne poskuse izolacije FHL2 iz inkluzijskih telesc in njihovo uspešnost primerjali med seboj. Pri vseh treh eksperimentih so se največje izgube pojavile pri procesu raztapljanja, po katerem je rekombinantni protein FHL2 ostal v obliki topnih agregatov, kljub prisotnosti denaturanta. Najobetavnejše rezultate je sicer pokazala izolacija, pri kateri smo pri procesu raztapljanja inkluzijskih telesc v 8 M urei dodali še neionski detergent Triton X-100, ki preprečuje tvorbo agregatov. Kljub znatnim količinam izoliranega fuzijskega proteina smo po dializi, pri kateri smo fuzijski protein cepili s proteazo TEV, ugotovili, da je FHL2 v raztopini podvržen razgradnji. Zaključili smo, da je izolacija rekombinantnega proteina FHL2 iz inkluzijskih telesc zaradi agregacije in nestabilnosti proteina nesmiselna. Ocenili smo, da bo bolj primerna metoda za pripravo proteina hkratno izražanje z njegovim vezavnim partnerjem. To je tudi v skladu z vlogo proteina FHL2 v celici, kjer se ta kot adapterski protein praktično nikoli ne nahaja sam, temveč je vedno v kompleksu z ostalimi proteini. Pri izvedbi koekspresije, kjer sta se zapisa za FHL2 in β-katenin nahajala vsak na svojem plazmidu, sta se proteina izražala v nivoju, zelo daleč od stehiometrijskega. V nadaljevanju smo se zato odločili, da bomo koekspresijo izvedli z zapisom obeh proteinov pod svojim promotorjem T7 na enem plazmidu in na ta način pridobili stabilen kompleks FHL2-β-katenin, s pomočjo katerega bo možno preučevanje interakcijskih in strukturnih lastnosti celotnega kompleksa EpIC-FHL2-β-katenin.

Ključne besede

rekombinantni proteini;adapterski protein;FHL2;ß-katenin;celično signaliziranje;inkluzijska telesca;hkratno izražanje;diplomska dela;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.11 - Diplomsko delo
Organizacija: UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [P. Škrinjar]
UDK: 577.112(043.2)
COBISS: 1538397891 Povezava se bo odprla v novem oknu
Št. ogledov: 772
Št. prenosov: 247
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Optimisation of recombinant protein FHL2 expression
Sekundarni povzetek: FHL2 is an adapter protein that contains four and a half LIM domains, which mediate interactions with other proteins. Between LIM domains there are intrinsically disordered regions that enable bonding to broad range of different proteins. This is the reason why FHL2 is a part of many signalling pathways. For example: it plays an important role in cell signalling mediated by Epithelial Cell Adhesion Molecule (EpCAM), which is highly expressed in cancer cells. FHL2, as an adaptor protein, mediates assembly of multiprotein complex EpIC-FHL2-β-catenin that is formed in cytoplasm and transported into the nucleus, where it binds to transcriptional factor Lef-1 and promotes transcription of oncogenes that cause cell proliferation. It is not clear how the complex EpIC-FHL2-β-catenin assembles and its structure is also unkown. The goal of our research was to optimize expression of recombinant FHL2 and to prepare sufficient amount for characterization of structural and biochemical properties of EpIC-FHL2-β-catenin complex. After induction of FHL2 expression in bacterial cells it is found in an insoluble fraction and it needs to be isolated from inclusion bodies. We carried out three experiments trying to recover recombinant FHL2 from inclusion bodies and we estimated the success of isolation for each one of them. In all three cases, we observed the largest loss of FHL2 due to unsuccessful solubilization of inclusion bodies, where our protein remained in the form of soluble aggregates, despite the presence of denaturant. We observed the most promising results when we add non-ionic detergent Triton X-100, that prevents formation of agreggates, to the process of inclusion bodies dissolution in 8 M urea. Even though we were able to recover substantial amount of fusion protein, we observed its unstability and degradation during the process of dialysis and proteolysis with TEV protease. We concluded that the isolation of recombinant FHL2 from inclusion bodies is pointless and is thus more suitable to preform coexpression of FHL2 with its binding partner. This method is also in line with the role of FHL2 protein in a cell, where it is present as an adapter protein and is therefore always present in a multiprotein complex. On top of that it also contains disordered regions between LIM domains, which is why it is prone to aggregation. To continue our work we would need to preform coexpression from one plasmid that would code for both proteins, FHL2 and β-catenin, each one with its own T7 promoter. If we carry out coexpression of FHL2 and β-catenin from different vectors, we get unequal expression of both proteins. With stable FHL2-β-catenin complex it would be possible to study interaction and structural properties of whole EpIC-FHL2-β-catenin complex.
Sekundarne ključne besede: FHL2;inclusion bodies;coexpression;ß-catenin;
Vrsta dela (COBISS): Diplomsko delo/naloga
Študijski program: 1000371
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Strani: 57 str.
ID: 11220299