Optmization of the α-Synuclein Antibody Stability through the Isoelectric Point for Parkinson's Disease Applications

Recent studies show that the aggregated and misfolded proteins within the brain directly and indirectly kill neurons, primarily through fibril formation one being α-Synuclein (αS). Studies of brains afflicted by Parkinsonâ??s disease have shown that protein fibrils form years before the disease actually is present. Utilizing E. coli bacterial expression, an antibody fragment was isolated that targets anti-α-Synuclein. This fraction is called a single chain variable fragment (scFv). It is easily produced, but does not have long-term stability. The primary goal of this investigation will be to determine new dialysis conditions that will ensure better stability of the scFv. Applying the ideal dialysis method could potentially create a more stable protein. Identifying an ideal isoelectric point through dialysis will benefit the overall stability of the structure.

This process starts with expressing the αS and then filtering the supernatant with the tangential flow filter (TFF). Afterward, the scFv is purified with ion metal affinity chromatography, utilizing nickel bead. The scFv bound to the nickel bead column, and is then released with the imidazole. Subsequently, the purified fraction is dialyzed in a variety stable storage buffers, including PBS, Tris, and HEPES. For each solution, the pH is adjusted to determine the effect on the stability of the purified fragments. The primary goal was to determine the isoelectric point, the settings where the proteins possess the equal amount of positive and negative charges. The electrostatic attraction contained in the protein can cause aggregation and produce precipitates, therefore rendering the engineered antigens useless for further study, therefore process conditions were accounted for throughout the study. An SDS-PAGE gel and western blot are used for confirmation of the identity of the protein, and a BCA test is applied determine protein concentration. Then, enzyme immunoassays (ELISA) are utilized to determine the stability and functionality of the scFv purified from the various buffers. The accumulated results of the ELISA were then compared to determine the most ideal dialysis conditions for anti-αS. This outcome became a standard procedure for future production of this particular protein.

After the series of analytical tests to confirm identity, functionality, and concentration, the results of the project concluded that phosphate-buffered saline at pH 7.4 provided the highest yield of scFv. Further studies must focus on the improvement of the stability of α-Synuclein through by identifying and improving laboratory protocol.