Combining Cell-Free Protein Synthesis with Coarse Grain Simulation to Discover Optimal Locations for Pegylation

The rapid production capability and open nature of cell-free systems can expedite design-build-test-learn cycles in protein engineering. Analogously, the computationally-efficient course-grain-molecular simulation method has emerged as a rapid in silico approach to also expedite protein engineering design cycles. Here we report applying course grain simulation to PEGylated proteins and combining course grain simulation results with cell-free protein synthesis to streamline design-build-test-learn cycles. These techniques enabled us to identify the locational impact of PEGylation on a model protein for retained stability and activity. In our initial case study, traditional design heuristics were insufficient to find the best PEGylation location. Thus, a combined simulation and experimental screen to facilitate rapid characterization of PEGylated protein variants has the potential to direct the creation of higher potency PEGylated protein therapeutics.

Reference: Wilding KM, Smith AK, Wilkerson JW, Bush DB, Knotts TA, Bundy BC. 2018.The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-free Protein Expression and Coarse-grain Simulation. ACS Synthetic Biology. 7(2):510-521