Electroporated recombinant proteins as tools for in vivo functional complementation, imaging, and chemical biology
- Max Planck Institute of Molecular Physiology, Germany
- Leibniz Institute of Molecular Pharmacology, Germany
- Max Planck Society, Germany
Abstract
Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and syntetic biology.
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All data generated or analysed during this study are included in the manuscript and supporting files
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Author details
Funding
Max-Planck-Gesellschaft (Open-access funding)
- Beate Voss
- Sabine Wohlgemuth
- Stefano Maffini
European Research Council (669686)
- Amal Alex
- Valentina Piano
- Soumitra Polley
- Giuseppe Ciossani
- Katharina Overlack
- Beate Voss
- Sabine Wohlgemuth
- Arsen Petrovic
- Andrea Musacchio
- Stefano Maffini
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Alex et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Electroporated recombinant proteins as tools for in vivo functional complementation, imaging, and chemical biology
eLife 8:e48287.
https://doi.org/10.7554/eLife.48287
