(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

(314a) Beginning with the End in Mind: Integrating Cell-Free Protein Synthesis with Coarse-Grained Simulation for Site-Specific Optimization of Protein Therapeutic Pegylation.


Bundy, B. - Presenter, Brigham Young University
Zhao, E., Brigham Young University
Soltani, M., Brigham Young University
Smith, A. K., Brigham Young University
Hunt, J. P., Brigham Young University
Wilkerson, J. W., Brigham Young University
Knotts, T. IV, Brigham Young University
Problem in Perspective: PEGylation is the only FDA-approved polymer to attach to therapeutic proteins for enhanced pharmacokinetics. However, PEGylation often comes at a high price where most of the protein therapeutic activity is lost (e.g. only 7% of the activity is retained when Interferon alpha is PEGylated1). This is due to an inability to perform site-specific PEGylation optimization due to the repeating nature of amino acids in proteins. For example, the commonly targeted primary amine of lysine is typically exposed on multiple locations of a protein and thus PEG is attached at different locations at different efficiencies depending on accessibility. Even with a significant setback in activity, PEGylated protein therapeutics are often more effective due to the longer retention times. However, there is significant potential to obtain enhanced pharmacokinetic retention with PEGylation and retain or even enhance protein therapeutics activity with location-optimized PEGylation. Due to the many variables at play and the inaccuracy of design heuristics, an integrated rapid simulation and high-throughput experimental approach is required.

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.


1) Bailon P, Palleroni A, Schaffer CA, Spence CL, Fung WJ, Porter JE, Ehrlich GK, Pan W, Xu ZX, Modi MW, Farid A. Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon α-2a for the treatment of hepatitis C. Bioconjugate chemistry. 2001 Mar 21;12(2):195-202.

2) Zhao EL, Soltani M, Smith AK, Hunt JP, Knotts IV TA, Bundy BC. Assessing Site-specific PEGylation of TEM-1 β-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 β-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.