De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37–43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.

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We thank M. Levitt and M. Zhang for discussions, A. Ford for data analysis advice, and Rosetta@Home participants for donating computing time. D.-A.S. thanks T. J. Brunette, J. E. Hsu and M. J. Countryman for their assistance. R.J. thanks K. Perry for X-ray data collection. We acknowledge funding support from: Life Sciences Discovery Fund Launch grant 9598385 (A.C.); PEW Latin-American fellow in the biomedical sciences and a CONACyT postdoctoral fellowship (D.-A.S.); Merck fellow of the Life Sciences Research Foundation (G.J.R.); CONACyT and Doctorado en Ciencias Bioquímicas UNAM (R.V.); NIH (R56AI117675) and Molecular Basis of Viral Pathogenesis Training Grant (T32AI007354-26A1) (S.M.B.); Investigator in the Pathogenesis of Infectious Disease award from the Burroughs Wellcome Fund and NIH (1R01NS080833) (M.D.); CoMotion Mary Gates Innovation Fellow program (T.C.); generous gift from Rocky and Genie Higgins (C.B.); Shenzhen Science and Technology Innovation Committee (JCYJ20170413173837121), Hong Kong Research Grant Council C6009-15G and AoE/P-705/16 (X.H.); PAPIIT UNAM (IN220516), CONACyT (254514) and Facultad de Medicina UNAM (D.A.F.-V.); NIAID grants (AI091823, AI123920, and AI125704) (R.J.); NIAID grant 1R41AI122431 (M.T.K. and D.H.F.); NIAID grant 1R21AI119258 and Life Sciences Discovery Fund grant 20040757 (D.H.F.). We acknowledge computing resources provided by the Supercomputing Laboratory at King Abdullah University of Science and Technology and the Hyak supercomputer system funded by the STF at the University of Washington. The Berkeley Center for Structural Biology is supported in part by the NIH, NIGMS, and HHMI. The Advanced Light Source is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. The Northeastern Collaborative Access Team beamlines are funded by NIGMS grant P41 GM103403 and a NIH-ORIP HEI grant (S10OD021527). Advanced Photon Source is a U.S. DOE Office of Science User Facility operated by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.

Author information

Author notes

    • Aaron Chevalier
    • , Daniel-Adriano Silva
    •  & Gabriel J. Rocklin

    These authors contributed equally to this work.


  1. Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA

    • Aaron Chevalier
    • , Daniel-Adriano Silva
    • , Gabriel J. Rocklin
    • , Derrick R. Hicks
    • , Renan Vergara
    • , Christopher D. Bahl
    • , Inna Goreshnik
    • , Tom Colvin
    • , Lauren Carter
    • , Cassie M. Bryan
    •  & David Baker
  2. Institute for Protein Design, University of Washington, Seattle, Washington 98195, USA.

    • Aaron Chevalier
    • , Daniel-Adriano Silva
    • , Gabriel J. Rocklin
    • , Derrick R. Hicks
    • , Renan Vergara
    • , Christopher D. Bahl
    • , Lauren Carter
    • , Cassie M. Bryan
    • , Lance Stewart
    •  & David Baker
  3. Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195, USA

    • Derrick R. Hicks
  4. Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, México City 04510, Mexico

    • Renan Vergara
    •  & D. Alejandro Fernández-Velasco
  5. Department of Microbiology, University of Washington, Seattle, Washington 98109, USA

    • Patience Murapa
    • , James T. Fuller
    • , Merika T. Koday
    • , Cody M. Jenkins
    • , Alan Bohn
    •  & Deborah H. Fuller
  6. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA

    • Steffen M. Bernard
    •  & Ian A. Wilson
  7. The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA

    • Steffen M. Bernard
    •  & Ian A. Wilson
  8. State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China

    • Lu Zhang
  9. Department of Chemistry and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

    • Lu Zhang
    •  & Xuhui Huang
  10. Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA

    • Kwok-Ho Lam
    • , Guorui Yao
    •  & Rongsheng Jin
  11. Department of Urology, Boston Children’s Hospital, Boston, Massachusetts 02115, USA

    • Shin-Ichiro Miyashita
    •  & Min Dong
  12. Department of Microbiology and Immunobiology and Department of Surgery, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Shin-Ichiro Miyashita
    •  & Min Dong
  13. Virvio Inc., Seattle, Washington 98195, USA

    • Merika T. Koday


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A.C., D.-A.S., G.J.R., D.H.F. and D.B. designed the research; A.C., D.-A.S., and G.J.R. contributed equally; D.R.H., R.V., and P.M. contributed equally; A.C., D.-A.S., G.J.R., D.R.H., R.V., and C.D.B. designed proteins. A.C., D.-A.S., D.R.H., R.V., performed binding experiments; S.M.B. solved influenza co-crystal structures; P.M., M.T.K., A.B., C.M.J. and J.T.F. performed influenza experiments; L.Z. performed molecular dynamics simulations; K.-H.L. and G.Y. solved BoNT co-crystal structures; S.-I.M. performed BoNT neutralization assays; I.G. and C.M.B. prepared yeast and next generation sequencing; T.C. performed protease-resistance characterization; L.C. performed protein purification. All authors analysed data. D.A.F.-V., L.S., M.D., X.H., R.J., I.A.W., D.H.F. and D.B. supervised research. A.C., D.-A.S., G.J.R., D.R.H., D.H.F. and D.B. wrote the manuscript.

Competing interests

Authors declare competing interests: A.C., M.T.K., D.H.F. and D.B. are co-founders and stockholders of Virvio, Inc., a company that aims to develop the therapeutics described in this manuscript. A.C., D.-A.S., G.J.R., C.D.B. and D.B. are co-inventors on a U.S. provisional patent application (No. 62/471,637) that incorporates discoveries described in this manuscript.

Corresponding author

Correspondence to David Baker.

Reviewer Information Nature thanks G. Nabel and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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