Periplasmic Expression and Characterization of an Alpha-Synuclein Preformed Fibril-Specific Nanobody and Its Antigen in Escherichia coli W3110 and W3110 ∆OMPC

The abnormal accumulation of alpha-synuclein (α-syn) is a core pathogenic mechanism in Parkinson’s disease. The nanobody PFFNB2 inhibits α-syn pre-formed fibrils (α-syn PFF) but faces expression challenges due to the absence of a canonical disulfide bond. This study aimed to express PFFNB2 and α-syn in the periplasm of Escherichia coli (E. coli) W3110 and its ompC-deficient mutant (∆ompC). PFFNB2 was expressed with and without a Maltose Binding Protein (MBP) fusion tag, while α-syn was expressed tag-free. Periplasmic proteins were extracted via osmotic shock, purified using chromatography, and analyzed for molecular weight and expression yield. The amyloid fibril-forming capacity of α-syn was evaluated using a Thioflavin T assay, and apparent binding potency was assessed via ELISA. Results demonstrated successful cloning, expression, and purification of both proteins. MBP-PFFNB2 yielded higher expression yield than the MBP-free form. Although the ∆ompC mutant showed a lower nanobody expression yield than the wild-type strain, the MBP-PFFNB2 obtained from this mutant exhibited optimal activity and specifically bound to α-syn PFF (EC50 of 0.3445 nM). Regarding α-syn, the ∆ompC mutant provided a superior expression yield, and lactose induction significantly reduced the formation of toxic oligomeric forms. In conclusion, combining periplasmic expression, an MBP fusion tag, and the E. coli W3110 ∆ompC strain creates a potential system for producing PFFNB2 nanobodies with high anti-α-syn PFF activity.