Transactive response DNA binding protein 43 kDa (TAR DNA-binding protein 43), a highly conserved RNA- and DNA-binding protein, is involved in the regulation of RNA processing. It is predominantly localized in the nucleus, while the accumulation of cytoplasmic aggregates is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. TDP-43 consists of an N-terminal domain, two RNA-recognition domains, a C-terminal domain, a nuclear localization signal, and a nuclear export signal. In this thesis, we detected the dimerization of TDP-43 using the Tox and Lex screening systems, employing wild-type TDP-43 and its two deletion mutants. Sequences encoding TDP-43 and two deletion mutants, TDP-43ΔNTD and TDP-43ΔNTDΔRRM1, were amplified by PCR from existing plasmids, and variants with the fusion partner maltose-binding protein (MBP) added at the C-terminus were also prepared. The prepared constructs were inserted into the Tox and Lex systems, and fluorescence was monitored as a readout of potential dimerization of the test protein. The Tox system is activator-based, meaning that homodimerization of the test protein increases fluorescence. The opposite occurs in the Lex system, which is repressor-based, where homodimerization of the test protein decreases fluorescence. Full-length TDP-43 showed a statistically significant dimerization signal in both systems. For the deletion mutants TDP-43ΔNTD and TDP-43ΔNTDΔRRM1, results were inconsistent between the two systems: in the Tox system, weak dimerization was detected for both mutants, whereas no dimerization was detected in the Lex system. The addition of the fusion partner MBP to the C-terminus of the deletion mutants reduced the dimerization signal in mutants in both systems, while for TDP-43 the dimerization signal remained unchanged or increased. These results suggest that the N-terminal domain is important for TDP-43 dimerization.
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