A Vaccinia-based system for directed evolution of GPCRs in mammalian cells

Christoph Klenk (), Maria Scrivens, Anina Niederer, Shuying Shi, Loretta Mueller, Elaine Gersz, Maurice Zauderer, Ernest S. Smith, Ralf Strohner and Andreas Plückthun ()
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Christoph Klenk: University of Zurich
Maria Scrivens: Vaccinex, Inc., 1895 Mt. Hope Avenue, Rochester
Anina Niederer: University of Zurich
Shuying Shi: Vaccinex, Inc., 1895 Mt. Hope Avenue, Rochester
Loretta Mueller: Vaccinex, Inc., 1895 Mt. Hope Avenue, Rochester
Elaine Gersz: Vaccinex, Inc., 1895 Mt. Hope Avenue, Rochester
Maurice Zauderer: Vaccinex, Inc., 1895 Mt. Hope Avenue, Rochester
Ernest S. Smith: Vaccinex, Inc., 1895 Mt. Hope Avenue, Rochester
Ralf Strohner: MorphoSys AG
Andreas Plückthun: University of Zurich

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: Abstract Directed evolution in bacterial or yeast display systems has been successfully used to improve stability and expression of G protein-coupled receptors for structural and biophysical studies. Yet, several receptors cannot be tackled in microbial systems due to their complex molecular composition or unfavorable ligand properties. Here, we report an approach to evolve G protein-coupled receptors in mammalian cells. To achieve clonality and uniform expression, we develop a viral transduction system based on Vaccinia virus. By rational design of synthetic DNA libraries, we first evolve neurotensin receptor 1 for high stability and expression. Second, we demonstrate that receptors with complex molecular architectures and large ligands, such as the parathyroid hormone 1 receptor, can be readily evolved. Importantly, functional receptor properties can now be evolved in the presence of the mammalian signaling environment, resulting in receptor variants exhibiting increased allosteric coupling between the ligand binding site and the G protein interface. Our approach thus provides insights into the intricate molecular interplay required for GPCR activation.

Date: 2023
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DOI: 10.1038/s41467-023-37191-8

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