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

Author

Listed:
  • Derek A. Wong

    (Northwestern University
    Northwestern University
    Northwestern University)

  • Zachary M. Shaver

    (Northwestern University
    Northwestern University
    Northwestern University
    Northwestern University)

  • Maria D. Cabezas

    (Northwestern University
    Northwestern University
    Northwestern University)

  • Martin Daniel-Ivad

    (Broad Institute of MIT and Harvard
    Harvard University)

  • Katherine F. Warfel

    (Northwestern University
    Northwestern University
    Northwestern University)

  • Deepali V. Prasanna

    (Northwestern University
    Northwestern University
    Northwestern University)

  • Sarah E. Sobol

    (Northwestern University
    Northwestern University
    Northwestern University)

  • Regina Fernandez

    (Northwestern University
    Northwestern University
    Northwestern University)

  • Fernando Tobias

    (Northwestern University
    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
    Cornell University
    Cornell University)

  • Emily P. Balskus

    (Broad Institute of MIT and Harvard
    Harvard University
    Harvard University)

  • Ashty S. Karim

    (Northwestern University
    Northwestern University
    Northwestern University)

  • Michael C. Jewett

    (Northwestern University
    Northwestern University
    Northwestern University
    Stanford University)

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.

Suggested Citation

  • 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, 2025. "Characterizing and engineering post-translational modifications with high-throughput cell-free expression," Nature Communications, Nature, vol. 16(1), pages 1-18, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-60526-6
    DOI: 10.1038/s41467-025-60526-6
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    References listed on IDEAS

    as
    1. Thapakorn Jaroentomeechai & Jessica C. Stark & Aravind Natarajan & Cameron J. Glasscock & Laura E. Yates & Karen J. Hsu & Milan Mrksich & Michael C. Jewett & Matthew P. DeLisa, 2018. "Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery," Nature Communications, Nature, vol. 9(1), pages 1-11, December.
    2. Richard S. Ayikpoe & Chengyou Shi & Alexander J. Battiste & Sara M. Eslami & Sangeetha Ramesh & Max A. Simon & Ian R. Bothwell & Hyunji Lee & Andrew J. Rice & Hengqian Ren & Qiqi Tian & Lonnie A. Harr, 2022. "A scalable platform to discover antimicrobials of ribosomal origin," Nature Communications, Nature, vol. 13(1), pages 1-15, December.
    3. Andrew C. Hunt & Bastian Vögeli & Ahmed O. Hassan & Laura Guerrero & Weston Kightlinger & Danielle J. Yoesep & Antje Krüger & Madison DeWinter & Michael S. Diamond & Ashty S. Karim & Michael C. Jewett, 2023. "A rapid cell-free expression and screening platform for antibody discovery," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    4. Wan-Qiu Liu & Xiangyang Ji & Fang Ba & Yufei Zhang & Huiling Xu & Shuhui Huang & Xiao Zheng & Yifan Liu & Shengjie Ling & Michael C. Jewett & Jian Li, 2024. "Cell-free biosynthesis and engineering of ribosomally synthesized lanthipeptides," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    5. Jasmine M. Hershewe & Katherine F. Warfel & Shaelyn M. Iyer & Justin A. Peruzzi & Claretta J. Sullivan & Eric W. Roth & Matthew P. DeLisa & Neha P. Kamat & Michael C. Jewett, 2021. "Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
    6. Grant M. Landwehr & Jonathan W. Bogart & Carol Magalhaes & Eric G. Hammarlund & Ashty S. Karim & Michael C. Jewett, 2025. "Accelerated enzyme engineering by machine-learning guided cell-free expression," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
    7. Weston Kightlinger & Katherine E. Duncker & Ashvita Ramesh & Ariel H. Thames & Aravind Natarajan & Jessica C. Stark & Allen Yang & Liang Lin & Milan Mrksich & Matthew P. DeLisa & Michael C. Jewett, 2019. "A cell-free biosynthesis platform for modular construction of protein glycosylation pathways," Nature Communications, Nature, vol. 10(1), pages 1-13, December.
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