Optimization of Process Variables for Design Ankyrin Repeat Proteins in Alzheimer's Diagnostic Applications

When normally healthy proteins in the brain begin to malfunction, they become misfolded and aggregate into conglomerates. These aggregations, called fibrils, are unable to be processed by the brain and therefore collect and cause damage to the surrounding neurons. As current forms of treatment are symptom based, there is little to no early-stage intervention. The diseases are tragically irreversible leaving little that doctors can do to help their patients. The development of a diagnostic system that can isolate the precursors to the degenerative effects will aid both researchers and patients to begin to better combat the disease.

Exploration of the applications of design Ankyrin repeat proteins (DARPin) has been used to combat Alzheimerâ??s disease with protein engineering research. These particular proteins perform similarly to an antibody, and bind to a given antigen. DARPin are capable of being manipulated to have binding regions that target specific antigens. Previous studies have revealed that α-Synuclein protein aggregations within neural cells are related to these neurological diseases. Through application, the DARPin protein structures are capable of binding to these aggregation sites, producing diagnostic tests against these known antigens. The research presented herein focuses on optimizing processing conditions for production of α-Synuclein-targeting DARPin for use as a diagnostic in Alzheimerâ??s patients. Once the presence of proteins is verified, the concentrations are measured using bicinchoninic acid assay (BCA). Enzyme-linked immunosorbent assay (ELISA) are used to analyze the functionality of the proteins. ELISAs are performed by stacking multiple layers of proteins and antigens, and then ultimately testing their presence and binding activity, using luminol. Various brain tissue samples (mouse, goat, and human) are used as targeted transgenic tissue for characterization. The brain tissue was genetically altered to overexpress α-synuclein to determine whether the synthesized proteins will bind well to their precursors.

The research has revealed that DARPin is optimized for a consistent growing temperature, regardless of protein structure. Utilizing a denaturing compound before running the proteins through SDS-PAGE gels improve the transfer through the media and aided in the characterization of the specific protein structure.