Characterizing and engineering post-translational modifications with high-throughput cell-free expression

Derek A. Wong, Zachary M. Shaver, Maria D. Cabezas, Martin Daniel-Ivad, Katherine F. Warfel, Deepali V. Prasanna, Sarah E. Sobol, Regina Fernandez, Fernando Tobias, Szymon K. Filip, Sophia W. Hulbert, Peter Faull, Robert Nicol, Matthew P. DeLisa, Emily P. Balskus (), Ashty S. Karim () and Michael C. Jewett ()
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Derek A. Wong: Northwestern University
Zachary M. Shaver: Northwestern University
Maria D. Cabezas: Northwestern University
Martin Daniel-Ivad: Broad Institute of MIT and Harvard
Katherine F. Warfel: Northwestern University
Deepali V. Prasanna: Northwestern University
Sarah E. Sobol: Northwestern University
Regina Fernandez: Northwestern University
Fernando Tobias: Northwestern University
Szymon K. Filip: Northwestern University
Sophia W. Hulbert: Cornell University
Peter Faull: Northwestern University
Robert Nicol: Broad Institute of MIT and Harvard
Matthew P. DeLisa: Cornell University
Emily P. Balskus: Broad Institute of MIT and Harvard
Ashty S. Karim: Northwestern University
Michael C. Jewett: Northwestern University

Nature Communications, 2025, vol. 16, issue 1, 1-18

Abstract: Abstract Post-translational modifications (PTMs) are important for the stability and function of many therapeutic proteins and peptides. Current methods for studying and engineering PTMs are often limited by low-throughput experimental techniques. Here we describe a generalizable, in vitro workflow coupling cell-free gene expression (CFE) with AlphaLISA for the rapid expression and testing of PTM installing proteins. We apply our workflow to two representative classes of peptide and protein therapeutics: ribosomally synthesized and post-translationally modified peptides (RiPPs) and glycoproteins. First, we demonstrate how our workflow can be used to characterize the binding activity of RiPP recognition elements, an important first step in RiPP biosynthesis, and be integrated into a biodiscovery pipeline for computationally predicted RiPP products. Then, we adapt our workflow to study and engineer oligosaccharyltransferases (OSTs) involved in protein glycan coupling technology, leading to the identification of mutant OSTs and sites within a model vaccine carrier protein that enable high efficiency production of glycosylated proteins. We expect that our workflow will accelerate design-build-test-learn cycles for engineering PTMs.

Date: 2025
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DOI: 10.1038/s41467-025-60526-6

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