magistrsko delo
Mojca Hunski (Author), Miha Pavšič (Mentor), Brigita Lenarčič (Thesis defence commission member), Marko Dolinar (Thesis defence commission member)

Abstract

Protein kinaze so med ključnimi proteini, ki sodelujejo pri prenosu signalov v sesalskih celicah. Mednje uvrščamo več kot 500 različnih človeških protein kinaz. V našem magistrskem delu smo se osredotočili na izoobliko delta iz družine protein kinaz C (PKCδ), ki sodeluje pri regulaciji pomembnih celičnih procesov, kot so apoptoza, diferenciacija in proliferacija. Gre za približno 78 kDa velik protein, ki ga sestavljata N-končna regulatorna in C-končna katalitična regija, katere struktura v primeru PKCδ še ni določena. Ugotovljeno je bilo, da citosolni del epitelijske celične adhezijske molekule (EpCAM) interagira s katalitično regijo PKCδ in jo s tem inhibira. PKCδ smo želeli pripraviti za podrobnejše raziskovanje interakcij s proteinom EpCAM. Katalitično regijo PKCδ smo želeli izraziti v bakterijskih celicah, vendar pri tem nismo bili uspešni – rekombinantnega proteina nismo uspeli pridobiti v zadostnih količinah v topni obliki. Z uporabo bakulovirusnega sistema za izražanje proteinov v insektnih celicah smo poskusili izraziti tako katalitično regijo kot celotno PKCδ. Uspešno smo izrazili le celotno PKCδ, kar smo potrdili z analizo z masno spektrometrijo, vendar se kjub poskusom čiščenja z nikljevo afiniteno kromatografijo ter ionskoizmenjevalno kromatografijo nismo znebili nečistot v proteinskem vzorcu. Z metodami molekulskega kloniranja smo pripravili še fuzijske vključke katalitične regije ter celotne PKCδ s fluorescenčnim proteinom mCherry za nadaljnje raziskave lokalizacije PKCδ. Pridobljeni rezultati služijo kot osnova za nadaljnje raziskave oziroma za optimizacijo priprave PKCδ. Pripravljene fuzijske vključke pa lahko uporabimo za nadaljnje raziskovanje delovanja proteina in znotrajceličnega signaliziranja in vivo ali in vitro.

Keywords

encimi;delta protein kinaza C;katalitična regija;izražanje;rekombinantni proteini;biokemijska karakteriazcija;molekulsko kloniranje;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [M. Hunski]
UDC: 577.15(043.2)
COBISS: 1538499523 Link will open in a new window
Views: 637
Downloads: 199
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Other data

Secondary language: English
Secondary title: Preparation and characterization of protein kinase C delta type
Secondary abstract: Protein kinases are among the most important proteins in signal transduction in mammalian cells. There are more than 500 different human protein kinases. In this work, we focused on delta isotype of protein kinase C (PKCδ), which is involved in regulation of many cell processes, such as apoptosis, differentiation and proliferation. It is a 78 kDa protein, composed of N-terminal regulatory region and C-terminal catalytic region. The structure of PKCδ catalytic region has not been determined yet. It has been shown that the cytoplasmic tail of epithelial cell adhesion molecule (EpCAM) interacts with PKCδ catalytic region and therefore inhibits it. We wanted to prepare PKCδ for the purpose of further research on PKCδ-EpCAM interaction. We have expressed PKCδ catalytic region in bacterial cells, yet we did not acquire adequate amount of soluble recombinant protein. Using the baculovirus expression system, the expression of PKCδ catalytic region and full length protein was carried out in insect cells. We have successfully expressed full-length PKCδ, which we identified with mass spectrometry. However, we could not remove impurities from the sample using affinity chromatography and ion exchange chromatography. Using molecular cloning techniques, we have also prepared constructs of catalytic region and full-length protein in fusion with fluorescent protein mCherry for the purpose of further investigation of PKCδ. Our results serve as a basis for further research or optimization of PKCδ preparation, while fusion constructs can provide novel tools for research of mechanisms of PKCδ action and intracellular signalling in vivo and in vitro.
Secondary keywords: PKC[delta];catalytic region;expression;recombinant protein;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 70 str.
ID: 11310540