Bacterial Expression and Purification of Recombinant Human α-Synuclein for Parkinson’s Disease Modelling

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders and is classified as a synucleinopathy due to the abnormal accumulation and aggregation of α-synuclein. Recombinant α-synuclein is widely used to investigate its structural properties, aggregation behavior, and contribution to disease mechanisms. However, these studies require reproducible production of sufficiently pure and concentrated protein. This study aimed to establish and optimize a workflow for the bacterial expression and purification of recombinant human wild-type α-synuclein in Escherichia coli to obtain purified monomeric α-synuclein suitable for controlled generation of pre-formed fibrils for downstream Parkinson’s disease modelling experiments. Two plasmid variants were evaluated, and recombinant α-synuclein expression was confirmed using molecular and protein-based analyses. The TAT variant was selected for further experiments to reduce the risk of cysteine misincorporation during bacterial expression. Purification enabled recovery of highly pure monomeric α-synuclein, although at relatively low yield. Overall, this study established a functional workflow for recombinant α-synuclein production and identified key steps that require further optimization to improve purity, recovery and suitability for controlled fibrillization experiments.