diplomsko delo
Miha Razdevšek (Avtor), Marko Novinec (Mentor)

Povzetek

V naravi se večina proteinov nahaja v oligomerni, predvsem homodimerni ali homotetramerni obliki. To encimom omogoča zvišano lokalno koncentracijo aktivnih mest, kar pospeši pretvorbo substrata. Ta lastnost je pri večini proteinov evolucijsko ugodna in se ohranja. Še vedno pa obstajajo monomerni proteini, ki se spontano ne povezujejo v oligomerne komplekse. Eden teh je katepsin B, cisteinska proteaza z endo- in eksonukleazno aktivnostjo. V sklopu diplomske naloge smo želeli z racionalnim pristopom poiskati aminokislinske ostanke na proteinu, ki bi jih lahko mutirali in s tem omogočili oligomerizacijo naravno monomernega proteina. V prvem delu diplomskega dela smo s pomočjo bioinformatskih orodij in teoretično znanih podatkov identificirali aminokislinske ostanke zadnje površine, smiselne za spremembo z naključno saturacijsko mutagenezo. Izbrali smo tiste, ki so bili obrnjeni ven, torej iz interakcijske površine. V drugem delu smo želeli in silico določena potencialno mutagena mesta preveriti še in vitro. Z naključno saturacijsko mutagenezo smo uvedli mutacije na specifična mesta in preverili dimerizacijsko stanje s sistemom BACTH. Do homodimerizacije dveh prokatepsinov B je prišlo ob uvedbi mutacije Glu9Val. Do heterodimerizacije pa pri mutaciji mesta 15, kjer je bila na eni podenoti prisotna mutacija Pro15Gly, druga podenota je bil divji tip proteina. Na koncu smo za oba potencialna dimera in silico vizualizirali najbolj verjetno strukturo dimera. Prvi se je povezal prek spodnje, drugi pa prek zadnje površine proteina.

Ključne besede

proteinski inženiring;proteini;oligomerizacija proteinov;proteaze;diplomska dela;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.11 - Diplomsko delo
Organizacija: UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [M. Razdevšek]
UDK: 577.15(043.2)
COBISS: 171467779 Povezava se bo odprla v novem oknu
Št. ogledov: 80
Št. prenosov: 23
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Protein engineering of dimeric variants of human cathepsin B with combination of rational design and random mutagenesis
Sekundarni povzetek: In nature, most proteins are found in an oligomeric, mainly homodimeric or homotetrameric form. This allows enzymes to have an increased local concentration of active sites, which speeds up the conversion of the substrate. This feature is evolutionarily favourable for most proteins and is conserved. However, there are still monomeric proteins that do not spontaneously associate into oligomeric complexes. One of these is cathepsin B, a cysteine protease with endo- and exonuclease activity. As part of the thesis, we wanted to use a rational design to find amino acid residues on the protein that could be mutated and thus allow oligomerisation of a naturally monomeric protein. In the first part of the thesis, we used bioinformatics tools and theoretically known data to identify amino acid residues on the back surface that are reasonable to be modified by random saturation mutagenesis. We selected those that were facing out, i.e. from the interaction surface. In the second part of the study, we wanted to in vitro test the in silico identified potential mutagenic sites. We introduced site-specific mutations by random saturation mutagenesis and checked the dimerization state with the BACTH system. Homodimerization of two molecules of procathepsin B occurred when the Glu9Val mutation was introduced. Heterodimerisation occurred when the mutation at site 15 was introduced, where one subunit carried the Pro15Gly mutation, and the other subunit was a wild-type protein. Finally, we in silico visualised the most probable dimer structure for both candidate dimers. The first one interacted via the bottom and the second one via the back surface of the protein.
Sekundarne ključne besede: cathepsin B;oligomerization;protein engineering;Katepsini;Univerzitetna in visokošolska dela;
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: 32 str.
ID: 19912990