Transposon-Display of AI-designed binders enables manipulation of the proteome in human cells

Hoch, A.; Fandrey, C. I.; Coventry, B.; Virreira Winter, S.; Geyer, P. E.; Kempchen, T. N.; Ebert, T. S.; Hoelzel, M.; Baker, D.; Schmid-Burgk, J. L.

doi:10.1101/2025.05.11.653342

bioRxiv · 10.1101/2025.05.11.653342

Transposon-Display of AI-designed binders enables manipulation of the proteome in human cells

Hoch, A.·Fandrey, C. I.·Coventry, B.·Virreira Winter, S.·Geyer, P. E.·Kempchen, T. N.·Ebert, T. S.·Hoelzel, M.·Baker, D.·Schmid-Burgk, J. L.

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Abstract

Transposon-Display is a highly scalable screening method that links proteins to their encoding DNA during expression in E. coli via a mutant transposase. Leveraging this system, we identified AI-designed binders targeting four intracellular human proteins, saturation-mutagenized top candidates, and charge-balanced variants to improve compatibility with the intracellular environment while preserving target binding. The resulting neutral binders can be fused to EGFP allowing antibody-free intracellular staining. Expressing neutral binders in human cells as fusions with functional domains can drive small molecule-controlled protein aggregation and trigger proteasomal degradation of endogenous target proteins in living cells. Manipulating the proteome of living cells using libraries of AI-designed binders may provide a new avenue to screen for disease-relevant protein functions and large-scale functional data may help to refine protein design algorithms.

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Hoch, A., Fandrey, C. I., Coventry, B., Virreira Winter, S., Geyer, P. E., Kempchen, T. N., Ebert, T. S., Hoelzel, M., Baker, D., Schmid-Burgk, J. L.. 2025-05-15. Transposon-Display of AI-designed binders enables manipulation of the proteome in human cells. https://doi.org/10.1101/2025.05.11.653342

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