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Published September 21, 2018 | Supplemental Material + Accepted Version
Journal Article Open

Programmable protein circuits in living cells


Synthetic protein-level circuits could enable engineering of powerful new cellular behaviors. Rational protein circuit design would be facilitated by a composable protein-protein regulation system in which individual protein components can regulate one another to create a variety of different circuit architectures. In this study, we show that engineered viral proteases can function as composable protein components, which can together implement a broad variety of circuit-level functions in mammalian cells. In this system, termed CHOMP (circuits of hacked orthogonal modular proteases), input proteases dock with and cleave target proteases to inhibit their function. These components can be connected to generate regulatory cascades, binary logic gates, and dynamic analog signal-processing functions. To demonstrate the utility of this system, we rationally designed a circuit that induces cell death in response to upstream activators of the Ras oncogene. Because CHOMP circuits can perform complex functions yet be encoded as single transcripts and delivered without genomic integration, they offer a scalable platform to facilitate protein circuit engineering for biotechnological applications.

Additional Information

© 2018 American Association for the Advancement of Science. This is an article distributed under the terms of the Science Journals Default License. Received 5 March 2018; accepted 14 August 2018. We thank J. Markson, Y. Antebi, N. Nandagopal, and J. Ruan for technical assistance; A. Varshavsky, R. Deshaies, and P. Coffino for scientific input and advice; and R. Kishony, G. Seelig, J.G. Ojalvo, J. Markson, L. Potvin-Trottier, K. Frieda, R. Zhu, and A. Granados for critical feedback. The research was funded by DARPA (HR0011-17-2-0008, M.B.E.), the Gordon and Betty Moore Foundation (GMBF2809, M.B.E.), NIH (T32 GM07616, L.S.C.), and the Helen Hay Whitney Foundation (F1047, X.J.G.). M.B.E is a Howard Hughes Medical Institute investigator. Author contributions: X.J.G. conceived of the project. X.J.G., L.S.C., M.S.K., and M.B.E. designed experiments. X.J.G., L.S.C., and M.S.K. performed experiments. X.J.G., L.S.C., M.S.K., and M.B.E. analyzed data and did mathematical modeling. X.J.G., L.S.C., and M.B.E. wrote the manuscript, with input from all authors. Competing interests: All authors are inventors on U.S. patent application 62/619,001 ("A System for Programming Protein-level Circuits in Living Cells") submitted by Caltech. Data and materials availability: All DNA constructs are available from Addgene (www.addgene.org/depositing/75869/), and cell lines available from M.B.E. under a material transfer agreement with Caltech. The datasets generated and analyzed and the computer code used during the current study are available upon request from the corresponding author.

Attached Files

Accepted Version - nihms-1574655.pdf

Supplemental Material - aat5062_Gao_SM.pdf

Supplemental Material - aat5062_Gao_Table_S1.xlsx

Supplemental Material - aat5062s1.mov


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August 19, 2023
October 18, 2023