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Rapid biosynthesis of glycoprotein therapeutics and vaccines from freeze-dried bacterial cell lysates

Nature Protocols volume 18, pages 2374–2398 (2023)Cite this article

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Abstract

The advent of distributed biomanufacturing platforms promises to increase agility in biologic production and expand access by reducing reliance on refrigerated supply chains. However, such platforms are not capable of robustly producing glycoproteins, which represent the majority of biologics approved or in development. To address this limitation, we developed cell-free technologies that enable rapid, modular production of glycoprotein therapeutics and vaccines from freeze-dried Escherichia coli cell lysates. Here, we describe a protocol for generation of cell-free lysates and freeze-dried reactions for on-demand synthesis of desired glycoproteins. The protocol includes construction and culture of the bacterial chassis strain, cell-free lysate production, assembly of freeze-dried reactions, cell-free glycoprotein synthesis, and glycoprotein characterization, all of which can be completed in one week or less. We anticipate that cell-free technologies, along with this comprehensive user manual, will help accelerate development and distribution of glycoprotein therapeutics and vaccines.

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Fig. 1: Cell-free systems accelerate glycoprotein production.
Fig. 2: Constructing glycosylation pathways.
Fig. 3: Cell-free lysate preparation methods.
Fig. 4: Reproducibility and optimization of cell-free glycoprotein synthesis.
Fig. 5: Cell-free systems synthesize diverse glycoproteins.

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Data availability

The data discussed in this manuscript were generated as part of our previously published work22,23,24. Source data are provided with this paper.

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Acknowledgements

J.C.S. acknowledges support from NIH/NCI F32 Postdoctoral Fellowship 1F32CA250324-01 and American Cancer Society Postdoctoral Fellowship PF-20-143-01-LIB. T.J. acknowledges support from the European Molecular Biology Organization Postdoctoral Fellowship 336-2021. K.F.W. acknowledges support from the National Defense Science and Engineering (NDSEG) Fellowship Program (ND-CEN-013-096). M.C.J. acknowledges support from the David and Lucile Packard Foundation, the Camille Dreyfus Teacher-Scholar Program, the Defense Threat Reduction Agency Grants HDTRA1-15-10052, HDTRA-12-11-0038 and HDTRA-12-01-0004, the Army Research Office Grants W911NF-20-1-0195, W911NF-18-1-0200 and W911NF-16-1-0372, the Army Contracting Command Contract W52P1J-21-9-3023 and DARPA Grant W911NF-23-2-0039.

Author information

Author notes

  1. Michael C. Jewett

    Present address: Department of Bioengineering, Stanford University, Stanford, CA, USA

Authors and Affiliations

  1. Department of Chemistry & Sarafan ChEM-H, Stanford University, Stanford, CA, USA

    Jessica C. Stark

  2. Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark

    Thapakorn Jaroentomeechai

  3. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA

    Katherine F. Warfel, Jasmine M. Hershewe & Michael C. Jewett

  4. Center for Synthetic Biology, Northwestern University, Evanston, IL, USA

    Katherine F. Warfel, Jasmine M. Hershewe & Michael C. Jewett

  5. Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA

    Katherine F. Warfel, Jasmine M. Hershewe & Michael C. Jewett

  6. Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA

    Matthew P. DeLisa

  7. Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, USA

    Michael C. Jewett

  8. Simpson-Querrey Institute, Northwestern University, Chicago, IL, USA

    Michael C. Jewett

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Contributions

J.C.S., T.J., K.F.W. and J.M.H. wrote and edited the manuscript. J.C.S., M.P.D. and M.C.J. conceptualized the manuscript. M.P.D. and M.C.J. directed the research and edited the manuscript.

Corresponding authors

Correspondence to Jessica C. Stark, Matthew P. DeLisa or Michael C. Jewett.

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Competing interests

M.C.J. is a cofounder of SwiftScale Biologics, Stemloop, Inc., Design Pharmaceuticals, and Pearl Bio. M.P.D. has interests in Glycobia Inc. and Versatope Inc. M.P.D. and M.C.J. have an interest in SwiftScale Biologics. M.C.J.’s and M.P.D.’s interests are reviewed and managed by Northwestern University and Cornell University, respectively, in accordance with their conflict of interest policies.

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Nature Protocols thanks Nigel Forest Reuel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Stark, J. C. et al. Sci. Adv. 7, eabe9444 (2021): https://doi.org/10.1126/sciadv.abe9444

Jaroentomeechai, T. et al. Nat. Commun. 9, 2686 (2018): https://doi.org/10.1038/s41467-018-05110-x

Natarajan, A. et al. Nat. Chem. Biol. 16, 1062–1070 (2020): https://doi.org/10.1038/s41589-020-0595-9

Hershewe, J. M. et al. Nat. Commun. 12, 2363 (2021): https://doi.org/10.1038/s41467-021-22329-3

Warfel, K. F. et al. ACS Synth. Biol. 12, 95–107 (2023): https://doi.org/10.1021/acssynbio.2c00392

Source data

Source Data Fig. 3

Statistical source data for Fig. 3b,c and uncropped western blot for Fig. 3c.

Source Data Fig. 4

Statistical source data for Fig. 4c and uncropped western blots for Fig. 4b,d.

Source Data Fig. 5

Uncropped western blots.

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Stark, J.C., Jaroentomeechai, T., Warfel, K.F. et al. Rapid biosynthesis of glycoprotein therapeutics and vaccines from freeze-dried bacterial cell lysates. Nat Protoc 18, 2374–2398 (2023). https://doi.org/10.1038/s41596-022-00799-z

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