20.500.12556/RUL-161294 Molekulsko kloniranje, izražanje in izolacija človeškega proteina FHL1 Molecular cloning, expression and isolation of the human FHL1 protein FHL1 (angl. Four and a half LIM domains protein 1) je protein, ki ga gradijo štiri in pol domene LIM. Domeno LIM gradita 2 zaporedna motiva cinkovih prstov, ki ju koordinirata dva iona Zn$^{2+}$, vsak ion po en motiv. Proteini FHL1 sodelujejo pri interakcijah z receptorji, transkripcijskimi proteini, proteini signalnih poti in strukturnimi proteini. FHL1 sodeluje kot transkripcijski faktor in sporočevalna molekula v veliko signalnih poteh, kar je razlog, da je vključen v veliko bolezenskih stanj. Med najbolj zaskrbljujoča spadajo srčno-žilne bolezni, kar je bil v zadnjih letih povod za povečano raziskovanje FHL1 in interakcij z drugimi proteini, saj so te slabo poznane. Cilj našega diplomskega dela je bil ugotoviti, če lahko po isti metodi kot za soroden protein FHL2 izrazimo in očistimo FHL1 ter tako pripravimo aktivno obliko tega proteina, kar bi lahko uporabili pri nadaljevanju analiz interakcij FHL1 z drugimi proteini. Zapis za FHL1 smo vnesli v vektor pET-32b(+), ki je vseboval vključek His6-sfGFP-TEV-2 × GSS-BamHI, z metodo IVA. Sledilo je izražanje celotnega vključka ter čiščenje FHL1, ki je potekalo z uporabo nikljeve afinitetne kromatografije. Med čiščenjem smo sfGFP in heksahistidinsko oznako odcepili s proteazo TEV. Rezultati analize z NaDS-PAGE po čiščenju so pokazali, da postopek čiščenja ni bil optimalen, saj smo pred dializo verjetno dodali premalo proteaze TEV in zato ni prišlo do odcepitve FHL1 od His6 in sfGFP. FHL1 je bil dobro topen. Nismo imeli težav z agregacijo proteinov. Za večji uspeh pri čiščenju bi bilo vredno poskusiti dodatek večjih količin proteaze TEV med dializo. FHL1 (Four and a Half LIM Domains Protein 1) is a protein composed of four and a half LIM domains. Each LIM domain consists of two consecutive zinc finger motifs, coordinated by two Zn$^{2+}$ ions, with each ion coordinating one motif. These domains are involved in protein–protein interactions, which include receptors, transcriptional proteins, signaling pathway proteins, and structural proteins. FHL1 functions as a transcription factor and signaling molecule in many pathways, which is why it is implicated in numerous disease states. Among the most concerning are cardiovascular diseases, leading to increased research into FHL1 and its interactions with other proteins, as these are not well understood. The aim of our thesis was to determine if FHL1 can be expressed and purified using the same method as for the related protein FHL2, thereby preparing an active form of this protein for subsequent interaction analysis with other proteins. We inserted the FHL1 gene into the pET-32b(+) vector, which contained the His6-sfGFP-TEV-2 × GSS-BamHI insert, using the IVA method. This was followed by expression of the entire insert and purification of FHL1 using nickel affinity chromatography. During purification, the sfGFP and hexahistidine tags were cleaved off using TEV protease. The results of NaDS-PAGE analysis post-purification indicated that the purification process was not optimal. This was likely due to the insufficient amount of TEV protease added before dialysis, resulting in incomplete cleavage of FHL1 from His6 and sfGFP. FHL1 was well soluble. We did not have any issues with protein aggregation. To achieve better purification results, it would be worthwhile to try adding larger amounts of TEV protease during dialysis. FHL1 transkripcijski faktor srčno-žilne bolezni FHL1 transcription factor cardiovascular diseases true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2024-09-09 12:00:01 2024-09-09 12:00:10 2024-11-07 11:44:59 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 24203 214035971 Vidmar_Nik_-_Molekulsko_kloniranje,_izrazanje_in_izolacija_cloveskega_proteina_FHL1.pdf Vidmar_Nik_-_Molekulsko_kloniranje,_izrazanje_in_izolacija_cloveskega_proteina_FHL1.pdf 1 31078CC618383BBD44E797F946373C05 3c315711755279ed1d866eed5e075f328d1dcc221532c540c44ee2888b579ae1 44b06e3d-6e92-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=190194 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0