Kloniranje in izražanje proteina TDP-43 in njegovih mutantov v bakterijskih celicah

Sernc, Maruša (author), Župunski, Vera (mentor)

TAR DNA vezavni protein oz. TDP-43 je 414 aminokislin dolg predstavnik družine heterogenih jedrnih ribonukleoproteinov (hnRNP). Sestavljen je iz urejene N-končne domene, ki je ključna za tvorbo homodimerne oblike, v kateri se protein, v fizioloških pogojih, največkrat nahaja. Nato sledi zanka, ki predstavlja jedrni lokalizacijski signal, tej pa dve RNA-prepoznavni domeni RRM1 in RRM2. Konec proteinske strukture predstavlja neurejena C-končna domena, ki je običajno podvržena številnim mutacijam. Protein TDP-43 se pretežno nahaja v jedru, kjer sodeluje v vseh stopnjah procesiranja in regulacije RNA, kontrolira izražanje genov in nekodirajočih RNA, omogoča stabilnost in transport mRNA, hkrati pa posreduje tudi pri tvorbi stresnih granul, kjer se mRNA z vezavnimi proteini poveže v majhne, agregirane citoplazemske strukture. Slednje nastanejo kot odgovor evkariontskih celic na stres iz okolja, katere se na ta način lahko osredotočijo le na sintezo proteinov, ključnih za preživetje celice. Kopičenje TDP-34 v citoplazmi je značilno za patološko stanje nevronov, kar vodi v celično smrt in je značilno za nekatere nevrodegenerativne bolezni, med katere sodi amiotrofična lateralna skleroza (ALS). V diplomskem delu smo poskušali izolirati divji tip proteina TDP-43 in štiri njegove mutante. Po metodi IVA kloniranja smo pripravili rekombinantni vektor pMCSG7, v katerega smo vstavili zapis za TDP-43 skupaj z zapisom za maltoza vezavni protein (MBP). Slednji se je v prvem primeru nahajal na N-koncu zapisa za TDP-43, v drugem primeru pa na C-koncu. Po izražanju v prokariontskih celicah E. coli seva BL21[DE3], smo fuzijski protein izolirali s pomočjo nikljeve afinitetne kromatografije in ga nato očistili na heparinski koloni. Ugotovili smo, da je stabilna le fuzija z MBP na C-koncu, fuzijski protein z MBP na N-koncu je fragmentiral med oz. po izolaciji. Pripravili smo dovolj velike količine proteina TDP-43-MBP za nadaljnje agregacijske in interakcijske študije. Od mutiranih oblik smo izolirali TDP-43 z oznako 301/307, ki vsebuje le obe regiji RRM, MBP pa ima, enako kot divji tip, na C-koncu. V nadaljnjem delu bi bilo smiselno optimizirati postopek izolacije mutiranih oblik proteina in izvesti agregacisjke in interakcijske študije TDP-43 in njegovih mutantov, ki so relevantni za nevrodegenerativne bolezni.

• Language: Slovenian
• Keywords: TDP-43, amiotrofična lateralna skleroza, mutanti, izražanje proteina, izolacija proteina
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-140028
• COBISS.SI-ID: 129809667
• Publication date in RUL: 09.09.2022

Secondary language

• Language: English
• Title: Cloning and expression of protein TDP-43 and its mutants in prokaryotic cells

Abstract

TAR DNA binding protein or TDP-43 is a 414 amino acid long member of heterogeneous ribonucleoprotein family (hnRNP). Its structure consists of a well-folded N-terminal domain involved in homodimerization, the most common form of the protein under physiological conditions. This is followed by the loop representing the nuclear localization signal, and then two RNA recognition motifs, RRM1 and RRM2. At the end of the protein structure is the disordered C-terminal domain which is a site of many mutations. The TDP-43 is mostly localized in the nucleus where it participates in all phases of RNA processing and regulation, controls the expression genes and non-coding RNA, facilitates the stability and transport of mRNA, and mediates in stress granules assembly. Stress granules are small, aggregated cytoplasmic structures, composed of mRNA and its binding proteins. They appear as a response of a eukaryotic cell answer to environmental stress when they must repress the expression of proteins that are not essential for survival. The accumulation of TDP-43 in the cytoplasm is typical of pathological conditions in neurons leading to cell death and consequently to some neurodegenerative disease such as amyotrophic lateral sclerosis (ALS). We aimed to isolate the wild type of TDP-43 and four of its mutants. Using the IVA cloning method, we prepared the recombinant vector pMCSG7, into which we inserted the TDP-43 sequence together with the maltose binding protein (MBP) sequence. We prepared two different recombinant vectors, the first with MBP at the N-terminus and the second with MBP at the C-terminus of TDP-43. After expression in E. coli BL21[DE3] cells, we isolated the fusion protein by nickel affinity chromatography and purified it with a heparin column. The results showed that only the fusion with MBP at the C-terminus was stable, whereas the fusion protein with MBP at the N-terminus fragmented during or/and after isolation. We isolated sufficient amounts of protein TDP-43 – MBP for subsequent aggregation and interaction studies. We managed to isolate one mutant form, with the mutation at sites 301/307, which only contains both RRM regions, and has an MBP fusion at the C-terminal domain. In further experiments, we should optimize the isolation procedure for mutant protein forms and perform aggregation and interaction studies of TDP-43 and its mutants relevant to neurodegenerative diseases.

• Keywords: TDP-43, amyotrophic lateral sclerosis, mutants, protein expression, protein isolation