Kloniranje in izražanje deamidacijskih mutantov proteina TDP-43 v bakteriji Escherichia coli

Škerjanc, Ariana Žana (author), Rogelj, Boris (mentor)

TDP-43 je DNA- in RNA- vezavni protein, ki spada med visoko ohranjene heterogene jedrne ribonukleoproteine (hnRNP). Za nevrodegenerativni bolezni amiotrofično lateralno sklerozo (ALS) in frontotemporalno demenco (FTD) je značilno kopičenje proteina TDP-43 v obliki netopnih citoplazemskih agregatov. TDP-43 je predvsem jedrni protein, ki je sestavljen iz strukturirane N-končne domene, dveh visoko ohranjenih RNA-prepoznavnih motivov in strukturno neurejene C-končne domene, ki je nagnjena k strukturnim spremembam in razgradnji. V fizioloških pogojih ima pomembno vlogo pri regulaciji procesov metabolizma RNA. Deamidacija je reakcija, pri kateri se amidna skupina stranske verige asparagina ali glutamina odstrani in nastane aspartat oz. glutamat. Deamidacija asparagina je pogosta posttranslacijska modifikacija, ki lahko vpliva na stabilnost, topnost in funkcijo proteinov, kar bi lahko vplivalo na patološko obnašanje proteina TDP-43. V okviru eksperimentalnega dela smo pripravili vektorje z vključki, ki zapisujejo štiri različne deamidacijske mutante TDP-43. Mutacije smo uvedli z metodo kloniranja s sestavljanjem in vivo (IVA), nato pa smo rekombinantne proteine z avtoindukcijo izražali v bakterijskih celicah E. coli BL21[DE3]. Na koncu smo izražanje proteina TDP-43 pred in po indukciji preverili z NaDS-PAGE in prenosom western ter imunodetekcijo. Rezultati dela predstavljajo prvi korak k razumevanju vpliva deamidacijskih mutacij na strukturo in stabilnost proteina TDP-43, prav tako pa pripravljeni mutanti omogočajo nadaljnje študije na področju patogeneze TDP-43.

• Language: Slovenian
• Keywords: TDP-43, deamidacija, izražanje proteina, molekulsko kloniranje
• Work type: Bachelor thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-186938
• Publication date in RUL: 07.09.2026

Secondary language

• Language: English
• Title: Cloning and expression of TDP-43 protein deamidation mutants in Escherichia coli bacteria

Abstract

TDP‑43 is a DNA‑ and RNA‑binding protein that belongs to highly conserved heterogeneous nuclear ribonucleoprotein (hnRNP) family. In the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), TDP‑43 accumulates in the form of insoluble cytoplasmic aggregates, which represent a key pathological hallmark. TDP‑43 is a primarily nuclear protein composed of a structured N‑terminal domain, two highly conserved RNA‑recognition motifs, and an intrinsically disordered C‑terminal domain that is prone to structural rearrangements and proteolytic cleavage. Under physiological conditions, TDP‑43 plays a key role in regulating of RNA metabolism. Deamidation, the conversion of asparagine or glutamine into aspartate or glutamate, is a common post‑translational modification that can alter protein stability, solubility, and function. These changes may influence the pathological behaviour of TDP‑43. In the experimental part of the study, we generated four deamidation mutants of TDP‑43 using in vivo assembly (IVA) cloning. The recombinant proteins were expressed in Escherichia coli BL21[DE3] using autoinduction, and protein expression before and after induction was analysed with NaDS‑PAGE and western blotting. The results confirm thrsuccessful preparation and expression of all four deamidated TDP‑43 mutants, representing an important first step toward understanding how deamidation may influence the structure and stability of TDP‑43. These mutants provide a valuable foundation for future studies of TDP-43 pathogenesis.

• Keywords: TDP‑43, deamidation, protein expression, molecular cloning