(523f) Location-Dependent Effect of Post-Translational Pegylation on Pegylation Efficiency and the Activity and Protein Stability of T4 Lysozyme

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 proteins’s molecular weight, thereby reducing clearance from the body. PEGylation also shields proteins from proteases and can thermodynamically stabilize a protein’s conformation. Thus PEGylation can also enhance protein biocatalysis stability and function. However, a central limitation to polymer-protein conjugation is polymer additions often 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 developed a rapid system to compare PEGylation location on model protein T4-lysozyme’s activity and stability. We also assess the impact of different PEG sizes, the accessibility of different locations for PEGylation, and the impact of PEGylation at multiple sites. The results of this study are compared to current design heuristics, simulations, and predictive tools for PEGylation. Overall, it is still challenging to predict a prior the optimal locations for PEGylation, however, additional structure-based guidelines to aid in the rational selection of optimal PEGylation sites are presented. Furthermore, the rapid screening system presented provides quick assessment of rationally selected PEGylation sites.