Rationally Engineering Post-Translational Protein Pegylation for Improved Stability and Function

Years of work conjugating the biocompatible polymer polyethylene glycol (PEG) to proteins have already resulted in the development of FDA-approved PEGylated protein therapeutics that are more stable and have longer functional lifetimes than their un-PEGylated counterparts. The post-translational covalent attachment of PEG groups, or PEGylation, increases a drug’s molecular weight, thereby reducing clearance from the body. PEGylation also shields the protein from proteases and can thermodynamically stabilize a protein’s conformation. Polymer-protein conjugation is a challenging task, however; polymer additions may interfere with protein folding and disrupt the protein or enzyme function. Thus, the attachment site of PEG on a protein must be optimized to achieve maximum stability and activity. Using cell-free protein synthesis and the incorporation of non-canonical amino acids, we examine the optimal conditions for conjugating PEG to proteins and compare the effect of PEGylation at different sites. This work will provide a basis for the development of structure-based guidelines to aid in the rational selection of optimal PEGylation sites.