20.500.12556/RUL-130169 Kloniranje in optimizacija izražanja rekombinantnih proteinov skupkov Neat1 in G4C2 za in vitro opazovanje njihovega nastanka Cloning and optimization of expression of recombinant proteins of Neat1 and G4C2 clusters for in vitro observation of their formation Prepisana heksanukleotidna razširitvena mutacija gena C9orf72 predstavlja osnovo za izgradnjo toksičnih nemembranskih skupkov G4C2. Te se pogosto pojavljajo pri družinskih različicah amiotrofične lateralne skleroze in frontotemporalne demence. Mehanizmi nastanka toksičnih skupkov niso dobro poznani in bi lahko bili temelj novega načina zdravljenja bolezni. Za raziskave skupkov so potrebni proteini, ki se kolokalizirajo z mutiranim prepisom in tvorijo skupke G4C2. Zato smo v okviru diplomske naloge pripravili plazmide za in vitro transkripcijo/translacijo in za izražanje proteinov SFPQ, NONO, PABPC1, TDP-43 ter FUS v bakterijah E. coli BL21(DE3). Nemutiranim različicam proteinov smo dodali fuzijski maltoza vezavni protein za povečano topnost konstruktov in fluorescenčni protein NeonGreen. Plazmid je vseboval tudi heksahistidinsko oznako in cepitveno mesto TEV za lažjo izolacijo konstruktov. Proteine SFPQ, PABPC1, TDP-43 in FUS smo izražali pri 37 oC, 1 mM IPTG, tri ure in pri 30 oC, 0,5 mM IPTG, pet ur. Najboljši pogoji za izražanje proteina so bili pri nižji temperaturi in nižji koncentraciji induktorja, ker se je takrat večina proteina nahajala v topni frakciji. V prenizkih količinah za nadaljnjo izolacijo sta se izražala konstrukta SFPQ in SFPQNeonGreen. V nadaljevanju je potrebno preveriti izražanje konstruktov NONO, NONONeonGreen in NeonGreen-PABPC1 ter z uporabo Ni2+ ionov izolirati izražene proteine, da dosežemo naš cilj. C9orf72 hexanucleotide repeat expansion transcript is the foundation on which toxic membraneless G4C2 foci are built. These are common among patients with familial amyotrophic lateral sclerosis and frontotemporal dementia. Mechanisms underlying the formation of toxic G4C2 foci are unknown and could represent the basis for new treatments. To research G4C2 foci proteins that localize and form these structures are needed. For that reason, this work focuses on the production of plasmids for in vitro transcription/translation and expression of SFPQ, NONO, PABPC1, TDP-43 and FUS proteins in E. coli BL21(DE3) bacteria. A fluorescent NeonGreen protein was fused to the wild type proteins along with maltose binding protein for increased construct solubility. The plasmids also contained a hexahistidine tag and a TEV cleavage site to assist with protein purification. SFPQ, PABPC1, TDP-43 and FUS were expressed at 37 oC with 1 mM IPTG for three hours and at 30 oC, with 0,5 mM IPTG for five hours. Lower temperature expression yielded a better result since most of the protein was in the soluble fraction of the cell lysate. Both SFPQ and SFPQ-NeonGreen were expressed in too low a quantity for further protein isolation. Further work needs to check the expression of NONO, NONO-NeonGreen, NeonGreen-PABPC1 and purify all of the constructs to achieve our goal. ALS FTD nemembranski organeli izražanje proteinov C9orf72 skupki G4C2 SFPQ TDP-43 NONO PABPC1 FUS ALS FTD membranelles organelles protein expression C9orf72 G4C2 foci SFPQ TDP-43 NONO PABPC1 FUS true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2021-09-10 15:35:00 2021-09-10 15:35:03 2022-09-09 04:15:08 0000-00-00 00:00:00 2021 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 17383 83269635 Nograsek_Teo_-_Kloniranje_in_optimizacija_izrazanja_rekombinantnih_proteinov_skupkov_Neat1_in_G4.pdf Nograsek_Teo_-_Kloniranje_in_optimizacija_izrazanja_rekombinantnih_proteinov_skupkov_Neat1_in_G4.pdf 1 795EFB5070DFB2C0B6A51C24E0D87755 64ccf89f8afab9ab18578a86fe0647823f85b8bc06a7ae5635745cebe8f1ed6e a7ac6df8-123b-11ec-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=147231 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0