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Irreversible light-activated SpyLigation mediates split-protein assembly in 4D

Nature Protocols volume 19, pages 1015–1052 (2024)Cite this article

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Abstract

The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide–protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly. LASL introduces an ortho-nitrobenzyl(oNB)-caged lysine into SC’s reactive site to generate a photoactivatable SC (pSC). Split-protein pairs of interest fused to pSC and ST are conditionally assembled via near-ultraviolet or pulsed near-infrared irradiation, as the uncaged SC can react with ST to ligate appended fragments. We describe procedures for the efficient synthesis of the photocaged amino acid that is incorporated within pSC (<5 days) as well as the design and cloning of LASL plasmids (1–4 days) for recombinant protein expression in either Escherichia coli (5–6 days) or mammalian cells (4–6 days), which require some prior expertise in protein engineering. We provide a chemoenzymatic scheme for appending bioorthogonal reactive handles onto E. coli-purified pSC protein (<4 days) that permits LASL component incorporation and patterned protein activation within many common biomaterial platforms. Given that LASL is irreversible, the photolithographic patterning procedures are fast and do not require sustained light exposure. Overall, LASL can be used to interrogate and modulate cell signaling in various settings.

Key points

  • The procedures cover a synthetic route to generate the photocaged lysine for photocaged SpyCatcher expression, the design of light-activated SpyLigation split proteins and plasmid vector design for expression in bacterial or mammalian cells.

  • The light-activated SpyLigation recombinant proteins are expressed and tested in solution, tethered into engineered biomaterials by site-specifically appending biorthogonal reactive handles on photocaged SpyCatcher through a sortase-mediated reaction, and transfected into mammalian cells for intracellular protein localization/activation via photolithographic patterning.

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Fig. 1: LASL affords complete spatiotemporal control over protein activation.
Fig. 2: In-solution assembly of UnaG and NanoLuc through LASL of split-protein fragments.
Fig. 3: LASL enables site-specific patterned protein localization and split-protein assembly in biomaterials.
Fig. 4: LASL-mediated split-protein activation in mammalian cells.
Fig. 5: Synthetic route and characterization of Lys(oNB).
Fig. 6: Workflow of LASL split-protein design.
Fig. 7: Schematic overview of the workflow for LASL E. coli protein expression.
Fig. 8: Schematic overview of the workflow for LASL biomaterial incorporation.
Fig. 9: Schematic overview of the workflow for LASL mammalian protein expression.

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Data availability

All pertinent experimental and characterization data discussed in this protocol are available in the supporting primary research articles11,26 and within this paper. Plasmids generated during the study are available on Addgene or from the corresponding author upon reasonable request.

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Acknowledgements

The authors recognize C. Butcher, T. Rapp, R. Francis, I. Kopyeva, R. Bretherton, M. Ross and N. Gregorio for feedback on the paper, as well as E. Ruskowitz, A. Strange, C. Butcher, S. Kurniawan and J. Filteau who further contributed to the original LASL manuscript. This work was supported by a CAREER Award (DMR 1652141 to C.A.D.) and grants (DMR 1807398 and CBET 1803054 to C.A.D.) from the National Science Foundation, as well as a Maximizing Investigators’ Research Award (R35GM138036 to C.A.D.) from the National Institutes of Health. Student fellowship support was provided by the Institute for Stem Cell and Regenerative Medicine (to B.G.M.-R.)

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Authors and Affiliations

  1. Department of Bioengineering, University of Washington, Seattle, WA, USA

    Brizzia G. Munoz-Robles & Cole A. DeForest

  2. Institute of Stem Cell & Regenerative Medicine, University of Washington, Seattle, WA, USA

    Brizzia G. Munoz-Robles & Cole A. DeForest

  3. Department of Chemical Engineering, University of Washington, Seattle, WA, USA

    Cole A. DeForest

  4. Department of Chemistry, University of Washington, Seattle, WA, USA

    Cole A. DeForest

  5. Molecular Engineering & Sciences Institute, University of Washington, Seattle, WA, USA

    Cole A. DeForest

  6. Institute for Protein Design, University of Washington, Seattle, WA, USA

    Cole A. DeForest

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Correspondence to Cole A. DeForest.

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Ruskowitz, E. R. et al. Nat. Chem. 15, 694–704 (2023): https://doi.org/10.1038/s41557-023-01152-x

Shadish, J. A. et al. Nat. Mater. 18, 1005–1014 (2019): https://doi.org/10.1038/s41563-019-0367-7

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Munoz-Robles, B.G., DeForest, C.A. Irreversible light-activated SpyLigation mediates split-protein assembly in 4D. Nat Protoc 19, 1015–1052 (2024). https://doi.org/10.1038/s41596-023-00938-0

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