Characterizing and engineering post-translational modifications with high-throughput cell-free expression
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...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-08-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-60526-6 |
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| author | 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 Michael C. Jewett |
| author_facet | 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 Michael C. Jewett |
| author_sort | Derek A. Wong |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-eeb37fe0049d4414bf7970db9866c7e4 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-eeb37fe0049d4414bf7970db9866c7e42025-08-20T04:03:01ZengNature PortfolioNature Communications2041-17232025-08-0116111810.1038/s41467-025-60526-6Characterizing and engineering post-translational modifications with high-throughput cell-free expressionDerek A. Wong0Zachary M. Shaver1Maria D. Cabezas2Martin Daniel-Ivad3Katherine F. Warfel4Deepali V. Prasanna5Sarah E. Sobol6Regina Fernandez7Fernando Tobias8Szymon K. Filip9Sophia W. Hulbert10Peter Faull11Robert Nicol12Matthew P. DeLisa13Emily P. Balskus14Ashty S. Karim15Michael C. Jewett16Department of Chemical and Biological Engineering, Northwestern UniversityChemistry of Life Processes Institute, Northwestern UniversityDepartment of Chemical and Biological Engineering, Northwestern UniversityBroad Institute of MIT and HarvardDepartment of Chemical and Biological Engineering, Northwestern UniversityDepartment of Chemical and Biological Engineering, Northwestern UniversityDepartment of Chemical and Biological Engineering, Northwestern UniversityDepartment of Chemical and Biological Engineering, Northwestern UniversityDepartment of Chemistry, Northwestern UniversityProteomics Center of Excellence, Northwestern UniversityBiochemistry, Molecular and Cell Biology (BMCB) Program, Cornell UniversityProteomics Center of Excellence, Northwestern UniversityBroad Institute of MIT and HarvardBiochemistry, Molecular and Cell Biology (BMCB) Program, Cornell UniversityBroad Institute of MIT and HarvardDepartment of Chemical and Biological Engineering, Northwestern UniversityDepartment of Chemical and Biological Engineering, Northwestern UniversityAbstract 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.https://doi.org/10.1038/s41467-025-60526-6 |
| spellingShingle | 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 Michael C. Jewett Characterizing and engineering post-translational modifications with high-throughput cell-free expression Nature Communications |
| title | Characterizing and engineering post-translational modifications with high-throughput cell-free expression |
| title_full | Characterizing and engineering post-translational modifications with high-throughput cell-free expression |
| title_fullStr | Characterizing and engineering post-translational modifications with high-throughput cell-free expression |
| title_full_unstemmed | Characterizing and engineering post-translational modifications with high-throughput cell-free expression |
| title_short | Characterizing and engineering post-translational modifications with high-throughput cell-free expression |
| title_sort | characterizing and engineering post translational modifications with high throughput cell free expression |
| url | https://doi.org/10.1038/s41467-025-60526-6 |
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