Molekulsko kloniranje, izražanje in izolacija človeškega proteina FHL3

Vujović, Nataša (avtor), Gaber, Aljaž (mentor)

FHL3 (angl. Four and a half LIM domain protein 3) je protein, ki je sestavljen iz štirih in pol domen LIM in se nahaja predvsem v skeletnih mišicah. Domene LIM koordinirajo cinkov ion med štiri ohranjene aminokislinske ostanke in so ključne kot interakcijski motivi med proteini. FHL3 preko protein–protein interakcij vpliva na izražanje določenih genov, diferenciacijo celic, oblikovanje citoskeleta in ima tudi vpliv na razvoj rakavih obolenj. Je eden od najmanj opisanih proteinov iz družine FHL. Interagira s transkripcijskimi faktorji, proteini, povezanimi s citoskeletom, in različnimi signalnimi molekulami. V okviru diplomskega dela smo želeli pripraviti topno obliko proteina FHL3 z uporabo enakega postopka, kot je bil uporabljen za pripravo topne oblike FHL2. V linearizirani vektor smo vstavili zapis za naš protein FHL3. FHL3 smo zaradi lažjega čiščenja in izolacije dodali fuzijska partnerja heksahistidinsko oznako (His6) in sfGFP, ki smo ju med čiščenjem odcepili s proteazo TEV. Za izražanje smo uporabili bakterijske celice E. coli seva BL21[DE3]. Protein smo uspešno izrazili, očistili in izolirali. Postopek, ki je bil uporabljen za pripravo topne oblike FHL2, se je izkazal kot učinkovit za pripravo topne oblike FHL3. Pripravljena topna oblika proteina FHL3 bi bila izjemno uporabna za nadaljnje raziskave njegove vloge v različnih celičnih procesih.

• Jezik: Slovenski jezik
• Ključne besede: FHL3, rekombinantni protein, domena LIM
• Vrsta gradiva: Diplomsko delo/naloga
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-161295
• Datum objave v RUL: 09.09.2024

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Molecular cloning, expression and isolation of the human FHL3 protein

Izvleček

FHL3 (Four and a half LIM domain protein 3) is a protein consisting of four and a half LIM domains found mainly in skeletal muscle. LIM domains coordinate the zinc ion between four conserved amino acid residues and are crucial as interaction motifs between proteins. FHL3 influences the expression of certain genes, cell differentiation, and cytoskeleton formation and has an impact on cancer development through protein–protein interactions. It is one of the least characterized proteins of the FHL family. It interacts with transcription factors, cytoskeleton-related proteins, and various signaling molecules. In the context of the thesis, we wanted to prepare a soluble form of FHL3 using the same procedure as was used to prepare a soluble form of FHL2. We inserted the transcript for our FHL3 protein into a linearised vector. To facilitate purification and isolation, we added to FHL3 fusion partners, hexahistidine tag (His6) and sfGFP, which were cleaved during purification by the TEV protease. For expression, E. coli strain BL21[DE3] bacterial cells were used. The protein was successfully expressed, purified, and isolated. The procedure used for the preparation of the soluble form of FHL2 proved to be effective for the preparation of the soluble form of FHL3. The prepared soluble form of FHL3 would be extremely useful for further research into its role in various cellular processes.

• Ključne besede: FHL3, recombinant protein, LIM domain