Synthesis of Functional Proteins via Bioconjugation
University of Delaware
Large protein synthesis is a present day biochemical challenge. Proteins can efficiently be expressed from various sources; however, post-translational modifications to introduce specific phosphorylation and spectroscopic labels can be challenging. One promising mechanism in creating these large, functional proteins is bioconjugation. This work is directed toward the chemical synthesis of microtubule-binding proteins and microtubule binding studies to elucidate the mechanism of tau aggregation in Alzheimer’s disease. Peptides from the tubulin-binding domain of Tau were synthesized on an automated peptide synthesizer and modifications were conducted, on both solid and solution phase, to allow incorporation of specific phosphorylation events and multiple bioorthogonal functionalities. Peptides were modified and peptide conjugation reactions were conducted. To elucidate the efficiency of these bioconjugation reactions we have used model peptides and demonstrated clean, efficient conversions in aqueous solutions. Further, we are utilizing these tools to synthesize proteins with multiple tubulin-binding domain repeats and to study their ability to bind, polymerize, and depolymerize microtubules in both their nonphosphorylated and phosphorylated states.