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National Repository of Grey Literature

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Optimization of p53 mutant protein isolation and its DNA binding properties Osadchuk, Olha ; Zemanová, Jana (referee) ; Brázda, Václav (advisor) Protein p53 je jednou z nejdůležitějších molekul v lidském těle. P53 reguluje celou řadu procesů v buňce, jako je například oprava DNA, buněčný cyklus nebo indukce apoptózy. Protein p53 je známý i jako „strážce genomu“. DNA vazebné schopnosti proteinu p53 jsou důležité pro normální vývoj a růst buňky. Mutace genu pro p53 mohou vést ke ztrátě jeho DNA vazebných vlastností a funkce nádorového supresoru, což muže způsobit rozvoj rakoviny. Teoretická část této diplomové práce je zaměřena na popis vlastností, funkce a mechanismus aktivace proteinu p53 a popis lokálních sekundárních struktur DNA. Hlavním cílem experimentální části byla produkce čtyř mutantních forem proteinů p53 a wild-type p53 proteinu a studium jejich vazebných vlastnosti s různými lokálními sekundárními strukturami DNA. Pomoci Gateway klonovacího systému byly připraveny čtyři expresní vektory, které byly použity pro produkci proteinů v bakteriálním expresním systému. Celkem byly úspěšně připraveny čtyři mutantní formy a wild-type p53 protein. Jejich vazebné vlastnosti byly studovány gelovou retardační analýzu. Výsledky naznačují různé DNA-vazebné vlastnosti wild-type p53 a studovaných mutantních forem tohoto proteinu. Všechny mutantní proteiny ztratily schopnost sekvenčně specificky vázat DNA, zatímco nespecifická interakce s DNA byla pozorována u tří ze čtyř mutantních forem. Jeden ze studovaných mutantních proteinů se vázal jenom na superhelikální formu DNA. Detailed record

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