Kloniranje in priprava delecijskih mutantov proteina TDP-43 v bakterijah Escherichia coli

Lah, Urša (avtor), Rogelj, Boris (mentor)

TDP-43 oz. protein z molekulsko maso 43 kDa, ki veže transaktivacijski odzivni element molekule DNA je predstavnik družine heterogenih jedrnih ribonukleoproteinov (hnRNP). TDP-43 je RNA- in DNA-vezavni protein, ki se v fizioloških pogojih pretežno nahaja v jedru. Tam sodeluje pri uravnavanju transkripcije in metabolizma RNA ter številnih drugih procesih, med katerimi so nadzorovanje izražanja genov, procesiranje miRNA, apoptoza, delitev celic in tvorba stresnih granul. Sestavljen je iz N-končne domene, dveh RNA-prepoznavnih motivov (RRM1 in RRM2) in C-končne domene. Poleg tega vsebuje tudi jedrni lokalizacijski signal in signal za transport iz jedra, ki omogočata premikanje TDP-43 med jedrom in citoplazmo. Kopičenje agregatov proteina TDP-43 in napačna lokalizacija so pogosti patološki znaki pri bolnikih z amiotrofično lateralno sklerozo (ALS) in frontotemporalno demenco (FTD). V diplomskem delu smo po metodi IVA-kloniranja ustvarili delecijske mutante proteina TDP-43, ki smo jih nato z avtoindukcijo uspešno izrazili v bakterijskih celicah Escherichia coli BL21[DE3]. Izražanje proteinov smo na koncu preverili z NaDS-PAGE in prenosom western.

• Jezik: Slovenski jezik
• Ključne besede: TDP-43, amiotrofična lateralna skleroza, molekulsko kloniranje, izražanje proteina
• Vrsta gradiva: Diplomsko delo/naloga
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-161274
• Datum objave v RUL: 09.09.2024

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Cloning and preparation of TDP-43 protein deletion mutants in Escherichia coli

Izvleček

Transactive response DNA binding protein 43 kDa, also known as TDP-43 is a representative of the heterogeneous nuclear ribonucleoprotein family (hnRNP). TDP-43 is an RNA- and DNA-binding protein that is under physiological conditions mainly located in the nucleus. There, it participates in the regulation of transcription and RNA metabolism, as well as many other processes, including the control of gene expression, miRNA processing, apoptosis, cell division and the formation of stress granules. It consists of N-terminal domain, two RNA-recognition motifs (RRM1 and RRM2) and C-terminal domain. In addition, it also contains a nuclear localization signal and a nuclear transport signal that enable shuttling of TDP-43 between the nucleus and the cytoplasm. Accumulation of TDP-43 protein aggregates and mislocalization are common pathological features in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We created deletion mutants of the TDP-43 protein using the IVA cloning method, which were then successfully expressed in Escherichia coli BL21[DE3] using autoinduction. Protein expression was examined with NaDS-PAGE and western blotting, whose results confirmed that we can continue the experimental work with isolation of the protein.

• Ključne besede: TDP-43, amyotrophic lateral sclerosis, molecular cloning, protein expression