(314a) Beginning with the End in Mind: Integrating Cell-Free Protein Synthesis with Coarse-Grained Simulation for Site-Specific Optimization of Protein Therapeutic Pegylation.
AIChE Annual Meeting
Tuesday, November 15, 2022 - 12:30pm to 12:48pm
Methods: Coarse-grained protein simulation is engineered to include PEG to allow rapid assessment of protein function and stability when PEG is theoretically attached at any and all locations on the target protein. Cell-free Protein Synthesis screening using noncanonical amino acids for site-specific covalent PEGylation of protein therapeutics enables high-throughput screening and validation of simulation results.
Results: Here we present the results of engineering coarse-grain protein simulation to include covalently attached PEG molecules and experimentally screening the impact of site-specific PEGylation using cell-free protein synthesis and noncanonical amino acids. These methods are applied to multiple protein targets with different sizes of PEG molecules. Remarkably, the coarse grain simulation is highly accurate in predicting the optimal location for PEGylation and the cell-free experimental method is able to rapidly verify the prediction. In this presentation we report our most recent results combined with recently published result from our labs2-5. Overall, this method has the potential to transform PEGylation. Rather than add PEG as an afterthought, in the future researchers will be able to optimize PEGylation at the beginning of protein therapeutic discovery and development. This would enable the development of more effective protein therapeutics with fewer administrations and side effects in the future.
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2) Zhao EL, Soltani M, Smith AK, Hunt JP, Knotts IV TA, Bundy BC. Assessing Site-specific PEGylation of TEM-1 Î²-lactamase with Cell-free Protein Synthesis and Coarse-grained Simulation. Journal of Biotechnology. 2022 Jan 5.
3) Smith AK, Soltani M, Wilkerson JW, Timmerman BD, Zhao EL, Bundy BC, Knotts IV TA. Coarse-grained simulation of PEGylated and tethered protein devices at all experimentally accessible surface residues on Î²-lactamase for stability analysis and comparison. The Journal of Chemical Physics. 2021 Feb 21;154(7):075102.
4) Wilkerson JW, Smith AK, Wilding KM, Bundy BC, Knotts IV TA. The Effects of p-Azidophenylalanine Incorporation on Protein Structure and Stability. Journal of Chemical Information and Modeling. 2020 Sep 23;60(10):5117-25.
5) Wilding KM, Smith AK, Wilkerson JW, Bush DB, Knotts IV TA, Bundy BC. The locational impact of site-specific PEGylation: streamlined screening with cell-free protein expression and coarse-grain simulation. ACS synthetic biology. 2018 Feb 16;7(2):510-21.