Site Selective Conjugation for Protein Therapeutics (2024)
Undergraduates: Benjamin Nguyen, Samantha Clouthier
Faculty Advisor: Wei You
Department: Chemistry
Protein therapeutics are an important route to drug delivery as they provide many advantages including high target specificity, few adverse side effects, and high tolerability by the body. However, there are many challenges for protein therapeutics, including low stability of proteins and rapid clearance of the proteins by the body, which ultimately decreases the efficacy of the protein therapeutic. One potential solution is the attachment of polymers to the protein through bioconjugation. Proteins can be delivered to the body in a controlled manner with the circulation time accounted for by controlling the size of the polymer they are bound to. In this investigation, bovine serum albumin (BSA) was utilized as a model protein as it has a single free thiol, allowing for site-specific conjugation through thiol maleimide chemistry for both a polymer and a small molecule linker with dibenzocyclooctyne (DBCO) functionality. Furthermore, the bioconjugation of BSA was characterized using various characterization techniques such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption ionization (MALDI), and thin layer chromatography (TLC) analysis using a fluorophore, which can be linked to the DBCO functionalized small molecule linker via click chemistry. Results from SDS-PAGE reveal the successful conjugation between BSA to both small molecule linker and polyethylene glycol (PEG) polymer. Likewise, TLC analysis demonstrated two consecutive conjugation reactions with small molecule linkers. However, MALDI indicated an unexpectedly large increase in molecular weight after conjugation. Further studies will be performed using various model proteins and ensuring bioconjugation between polymer and proteins.