CF2H: a cell-free two-hybrid platform for rapid protein binder screening

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cf2h:-a-cell-free-two-hybrid-platform-for-rapid-protein-binder-screening
CF2H: a cell-free two-hybrid platform for rapid protein binder screening

References

  1. Pawson, T. & Nash, P. Protein-protein interactions define specificity in signal transduction. Genes Dev. 14, 1027–1047 (2000).

    Google Scholar 

  2. Good, M. C., Zalatan, J. G. & Lim, W. A. Scaffold proteins: hubs for controlling the flow of cellular information. Science 332, 680–686 (2011).

    Google Scholar 

  3. Sahni, N. et al. Widespread macromolecular interaction perturbations in human genetic disorders. Cell 161, 647–660 (2015).

    Google Scholar 

  4. Ribas, A. & Wolchok, J. D. Cancer immunotherapy using checkpoint blockade. Science 359, 1350–1355 (2018).

    Google Scholar 

  5. Kortemme, T. De novo protein design-from new structures to programmable functions. Cell 187, 526–544 (2024).

    Google Scholar 

  6. Watson, J. L. et al. De novo design of protein structure and function with RFdiffusion. Nature 620, 1089–1100 (2023).

    Google Scholar 

  7. Dauparas, J. et al. Robust deep learning–based protein sequence design using ProteinMPNN. Science 378, 49–56 (2022).

    Google Scholar 

  8. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Google Scholar 

  9. Gainza, P. et al. De novo design of protein interactions with learned surface fingerprints. Nature 617, 176–184 (2023).

    Google Scholar 

  10. Bennett, N. R. et al. Improving de novo protein binder design with deep learning. Nat. Commun. 14, 2625 (2023).

    Google Scholar 

  11. Pacesa, M. et al. One-shot design of functional protein binders with BindCraft. Nature 646, 483–492 (2025).

    Google Scholar 

  12. Kim, G. et al. Easy and accurate protein structure prediction using ColabFold. Nat. Protoc. 20, 620–642 (2025).

    Google Scholar 

  13. Wohlwend, J. et al. Boltz-1 democratizing biomolecular interaction modeling. Preprint at https://doi.org/10.1101/2024.11.19.624167 (2024).

  14. Ovchinnikov, S. ColabDesign: Making Protein Design Accessible to All via Google Colab! https://github.com/sokrypton/ColabDesign (Github).

  15. Zhou, Y. et al. Engineering bacterial transcription regulation to create a synthetic in vitro two-hybrid system for protein interaction assays. J. Am. Chem. Soc. 136, 14031–14038 (2014).

    Google Scholar 

  16. Shimizu, Y. et al. Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).

    Google Scholar 

  17. Lavickova, B. & Maerkl, S. J. A simple, robust, and low-cost method to produce the PURE cell-free system. ACS Synth. Biol. 8, 455–462 (2019).

    Google Scholar 

  18. Sun, Z. Z. et al. Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology. J. Vis. Exp. 79, e50762 (2013).

  19. Garenne, D. et al. Cell-free gene expression. Nat. Rev. Methods Primers 1, 49 (2021).

  20. Wagner, L., Jules, M. & Borkowski, O. What remains from living cells in bacterial lysate-based cell-free systems. Comput. Struct. Biotechnol. J. 21, 3173–3182 (2023).

    Google Scholar 

  21. Foshag, D. et al. The E. coli S30 lysate proteome: a prototype for cell-free protein production. N. Biotechnol. 40, 245–260 (2018).

    Google Scholar 

  22. Hillebrecht, J. R. & Chong, S. A comparative study of protein synthesis in in vitro systems: from the prokaryotic reconstituted to the eukaryotic extract-based. BMC Biotechnol. 8, 58 (2008).

    Google Scholar 

  23. Batista, A. C. et al. Differentially optimized cell-free buffer enables robust expression from unprotected linear DNA in exonuclease-deficient extracts. ACS Synth. Biol. 11, 732–746 (2022).

    Google Scholar 

  24. Blaszczak, E., Lazarewicz, N., Sudevan, A., Wysocki, R. & Rabut, G. Protein-fragment complementation assays for large-scale analysis of protein-protein interactions. Biochem. Soc. Trans. 49, 1337–1348 (2021).

    Google Scholar 

  25. Kudla, J. & Bock, R. Lighting the way to protein-protein interactions: Recommendations on best practices for bimolecular fluorescence complementation analyses. Plant Cell 28, 1002–1008 (2016).

    Google Scholar 

  26. Romei, M. G. & Boxer, S. G. Split green fluorescent proteins: scope, limitations, and outlook. Annu. Rev. Biophys. 48, 19–44 (2019).

    Google Scholar 

  27. McSweeney, M. A. et al. A modular cell-free protein biosensor platform using split T7 RNA polymerase. Sci. Adv. 11, eado6280 (2025).

    Google Scholar 

  28. Ptashne, M. A Genetic Switch: Phage Lambda Revisited. (Cold Spring Harbor Laboratory Press, 2004).

  29. Pabo, C. O., Sauer, R. T., Sturtevant, J. M. & Ptashne, M. The lambda repressor contains two domains. Proc. Natl. Acad. Sci. USA 76, 1608–1612 (1979).

    Google Scholar 

  30. Sauer, R. T., Pabo, C. O., Meyer, B. J., Ptashne, M. & Backman, K. C. Regulatory functions of the lambda repressor reside in the amino-terminal domain. Nature 279, 396–400 (1979).

    Google Scholar 

  31. Weiss, M. A., Pabo, C. O., Karplus, M. & Sauer, R. T. Dimerization of the operator binding domain of phage lambda repressor. Biochemistry 26, 897–904 (1987).

    Google Scholar 

  32. Hu, J. C., O’Shea, E., Kim, P. S. & Sauer, R. Sequence requirements for coiled-coils: analysis with lambda repressor-GCN4 leucine zipper fusions. Science 250, 1400–1403 (1990).

    Google Scholar 

  33. Park, S. H. & Raines, R. T. Genetic selection for dissociative inhibitors of designated protein-protein interactions. Nat. Biotechnol. 18, 847–851 (2000).

    Google Scholar 

  34. Mariño-Ramírez, L., Campbell, L. & Hu, J. C. Screening peptide/protein libraries fused to the lambda repressor DNA-binding domain in E. coli cells. Methods Mol. Biol. 205, 235–250 (2003).

    Google Scholar 

  35. Brödel, A. K., Jaramillo, A. & Isalan, M. Engineering orthogonal dual transcription factors for multi-input synthetic promoters. Nat. Commun. 7, 13858 (2016).

    Google Scholar 

  36. Bushman, F., Shang, C. & Ptashne, M. A single glutamic acid residue plays a key role in the transcriptional activation function of lambda repressor. Cell 58, 1163–1171 (1989).

    Google Scholar 

  37. Woolfson, D. N. Coiled-coil design: updated and upgraded. Subcell. Biochem. 82, 35–61 (2017).

    Google Scholar 

  38. Lebar, T., Lainšček, D., Merljak, E., Aupič, J. & Jerala, R. A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells. Nat. Chem. Biol. 16, 513–519 (2020).

    Google Scholar 

  39. Pardee, K. et al. Portable, on-demand biomolecular manufacturing. Cell 167, 248–259.e12 (2016).

    Google Scholar 

  40. Götzke, H. et al. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat. Commun. 10, 4403 (2019).

    Google Scholar 

  41. Vázquez Torres, S. et al. De novo design of high-affinity binders of bioactive helical peptides. Nature 626, 435–442 (2024).

    Google Scholar 

  42. Wojcik, J. et al. A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain. Nat. Struct. Mol. Biol. 17, 519–527 (2010).

    Google Scholar 

  43. Keeble, A. H. et al. Approaching infinite affinity through engineering of peptide-protein interaction. Proc. Natl. Acad. Sci. USA 116, 26523–26533 (2019).

    Google Scholar 

  44. Brauchle, M. et al. Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry. Biol. Open 3, 1252–1261 (2014).

    Google Scholar 

  45. Fridy, P. C. et al. A robust pipeline for rapid production of versatile nanobody repertoires. Nat. Methods 11, 1253–1260 (2014).

    Google Scholar 

  46. Shui, S. et al. A rational blueprint for the design of chemically-controlled protein switches. Nat. Commun. 12, 5754 (2021).

    Google Scholar 

  47. Pascolutti, R. et al. Structure and dynamics of PD-L1 and an ultra-high-affinity PD-1 receptor mutant. Structure 24, 1719–1728 (2016).

    Google Scholar 

  48. Ellenberger, T. E., Brandl, C. J., Struhl, K. & Harrison, S. C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. Cell 71, 1223–1237 (1992).

    Google Scholar 

  49. Fletcher, J. M. et al. A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology. ACS Synth. Biol. 1, 240–250 (2012).

    Google Scholar 

  50. Jeffrey, P. D., Gorina, S. & Pavletich, N. P. Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 angstroms. Science 267, 1498–1502 (1995).

    Google Scholar 

  51. Zaccai, N. R. et al. A de novo peptide hexamer with a mutable channel. Nat. Chem. Biol. 7, 935–941 (2011).

    Google Scholar 

  52. Liu, J. et al. A seven-helix coiled coil. Proc. Natl. Acad. Sci. USA 103, 15457–15462 (2006).

    Google Scholar 

  53. Hendrickson, W. A. et al. Crystal structure of core streptavidin determined from multiwavelength anomalous diffraction of synchrotron radiation. Proc. Natl. Acad. Sci. USA 86, 2190–2194 (1989).

    Google Scholar 

  54. Ingram, J. R. et al. Localized CD47 blockade enhances immunotherapy for murine melanoma. Proc. Natl. Acad. Sci. USA 114, 10184–10189 (2017).

    Google Scholar 

  55. Mullard, A. 2024 FDA approvals. Nat. Rev. Drug Discov. 24, 75–82 (2025).

    Google Scholar 

  56. Holzer, P. et al. Discovery of a dihydroisoquinolinone derivative (NVP-CGM097): A highly potent and selective MDM2 inhibitor undergoing phase 1 clinical trials in p53wt tumors. J. Med. Chem. 58, 6348–6358 (2015).

    Google Scholar 

  57. Souers, A. J. et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat. Med. 19, 202–208 (2013).

    Google Scholar 

  58. Stanton, B. Z., Chory, E. J. & Crabtree, G. R. Chemically induced proximity in biology and medicine. Science 359, eaao5902 (2018).

  59. Scheller, L., Strittmatter, T., Fuchs, D., Bojar, D. & Fussenegger, M. Generalized extracellular molecule sensor platform for programming cellular behavior. Nat. Chem. Biol. 14, 723–729 (2018).

    Google Scholar 

  60. Guo, Z. et al. Generalizable protein biosensors based on synthetic switch modules. J. Am. Chem. Soc. 141, 8128–8135 (2019).

    Google Scholar 

  61. Chang, H.-J. et al. A modular receptor platform to expand the sensing repertoire of bacteria. ACS Synth. Biol. 7, 166–175 (2018).

    Google Scholar 

  62. Rihtar, E. et al. Chemically inducible split protein regulators for mammalian cells. Nat. Chem. Biol. 19, 64–71 (2023).

    Google Scholar 

  63. Quijano-Rubio, A. et al. De novo design of modular and tunable protein biosensors. Nature 591, 482–487 (2021).

    Google Scholar 

  64. Sonneson, G. J. & Horn, J. R. Hapten-induced dimerization of a single-domain VHH camelid antibody. Biochemistry 48, 6693–6695 (2009).

    Google Scholar 

  65. Lesne, J. et al. Structural basis for chemically-induced homodimerization of a single domain antibody. Sci. Rep. 9, 1840 (2019).

    Google Scholar 

  66. Wang, T. et al. Caffeine-operated synthetic modules for chemogenetic control of protein activities by life style. Adv. Sci. 8, 2002148 (2021).

    Google Scholar 

  67. Smith, C. A. et al. Molecular recognition requires dimerization of a VHH antibody. MAbs 15, 2215363 (2023).

    Google Scholar 

  68. Feng, J. et al. A general strategy to construct small molecule biosensors in eukaryotes. Elife 4, e10606 (2015).

  69. Anderson, N. L. The clinical plasma proteome: a survey of clinical assays for proteins in plasma and serum. Clin. Chem. 56, 177–185 (2010).

    Google Scholar 

  70. Zhang, J., Xiao, T., Cai, Y. & Chen, B. Structure of SARS-CoV-2 spike protein. Curr. Opin. Virol. 50, 173–182 (2021).

    Google Scholar 

  71. Koenig, P.-A. et al. Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape. Science 371, eabe6230 (2021).

  72. Borkowski, O. et al. Large scale active-learning-guided exploration for in vitro protein production optimization. Nat Commun 11, 1872 (2020).

  73. Hunt, A. C. et al. A rapid cell-free expression and screening platform for antibody discovery. Nat. Commun. 14, 3897 (2023).

    Google Scholar 

  74. Matsuda, T. et al. Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody-antigen interaction. PLoS ONE 13, e0193158 (2018).

    Google Scholar 

  75. Matsuda, T., Watanabe, S. & Kigawa, T. Cell-free synthesis system suitable for disulfide-containing proteins. Biochem. Biophys. Res. Commun. 431, 296–301 (2013).

    Google Scholar 

  76. Kim, D.-M. & Swartz, J. R. Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli. Biotechnol. Bioeng. 85, 122–129 (2004).

    Google Scholar 

  77. Burz, D. S. & Ackers, G. K. Single-site mutations in the C-terminal domain of bacteriophage lambda cI repressor alter cooperative interactions between dimers adjacently bound to OR. Biochemistry 33, 8406–8416 (1994).

    Google Scholar 

  78. Burz, D. S., Beckett, D., Benson, N. & Ackers, G. K. Self-assembly of bacteriophage lambda cI repressor: effects of single-site mutations on the monomer-dimer equilibrium. Biochemistry 33, 8399–8405 (1994).

    Google Scholar 

  79. Brenowitz, M., Senear, D. F. & Ackers, G. K. Flanking DNA-sequences contribute to the specific binding of cI-repressor and OR1. Nucleic Acids Res 17, 3747–3755 (1989).

    Google Scholar 

  80. Garamella, J., Marshall, R., Rustad, M. & Noireaux, V. The all E. coli TX-TL toolbox 2.0: a platform for cell-free synthetic biology. ACS Synth. Biol. 5, 344–355 (2016).

    Google Scholar 

  81. Capin, J., Chabert, E., Zuñiga, A. & Bonnet, J. Microbial biosensors for diagnostics, surveillance and epidemiology: today’s achievements and tomorrow’s prospects. Microb. Biotechnol. 17, e70047 (2024).

    Google Scholar 

  82. Jung, J. K. et al. Cell-free biosensors for rapid detection of water contaminants. Nat. Biotechnol. 38, 1451–1459 (2020).

    Google Scholar 

  83. Buzas, D. et al. In vitro generated antibodies guide thermostable ADDomer nanoparticle design for nasal vaccination and passive immunization against SARS-CoV-2. Antib. Ther. 6, 277–297 (2023).

    Google Scholar 

  84. Capin, J. et al. CF2H: a Cell-free two-hybrid platform for rapid protein binder screening. synthetic-biology-group-cbs-montpellier/CF2H: v1.0.0 https://doi.org/10.5281/zenodo.18504886 (2026).

  85. Broos, K. et al. Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers. Oncotarget 8, 41932–41946 (2017).

    Google Scholar 

  86. Discovery, C. et al. Chai-1: Decoding the molecular interactions of life. Preprint at https://doi.org/10.1101/2024.10.10.615955 (2024).

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