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Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology

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 sur...

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Autores principales: Alex, Amal, Piano, Valentina, Polley, Soumitra, Stuiver, Marchel, Voss, Stephanie, Ciossani, Giuseppe, Overlack, Katharina, Voss, Beate, Wohlgemuth, Sabine, Petrovic, Arsen, Wu, Yaowen, Selenko, Philipp, Musacchio, Andrea, Maffini, Stefano
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656429/
https://www.ncbi.nlm.nih.gov/pubmed/31310234
http://dx.doi.org/10.7554/eLife.48287
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author Alex, Amal
Piano, Valentina
Polley, Soumitra
Stuiver, Marchel
Voss, Stephanie
Ciossani, Giuseppe
Overlack, Katharina
Voss, Beate
Wohlgemuth, Sabine
Petrovic, Arsen
Wu, Yaowen
Selenko, Philipp
Musacchio, Andrea
Maffini, Stefano
author_facet Alex, Amal
Piano, Valentina
Polley, Soumitra
Stuiver, Marchel
Voss, Stephanie
Ciossani, Giuseppe
Overlack, Katharina
Voss, Beate
Wohlgemuth, Sabine
Petrovic, Arsen
Wu, Yaowen
Selenko, Philipp
Musacchio, Andrea
Maffini, Stefano
author_sort Alex, Amal
collection PubMed
description 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 synthetic biology.
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spelling pubmed-66564292019-07-25 Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology Alex, Amal Piano, Valentina Polley, Soumitra Stuiver, Marchel Voss, Stephanie Ciossani, Giuseppe Overlack, Katharina Voss, Beate Wohlgemuth, Sabine Petrovic, Arsen Wu, Yaowen Selenko, Philipp Musacchio, Andrea Maffini, Stefano eLife Biochemistry and Chemical Biology 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 synthetic biology. eLife Sciences Publications, Ltd 2019-07-16 /pmc/articles/PMC6656429/ /pubmed/31310234 http://dx.doi.org/10.7554/eLife.48287 Text en © 2019, Alex et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Biochemistry and Chemical Biology
Alex, Amal
Piano, Valentina
Polley, Soumitra
Stuiver, Marchel
Voss, Stephanie
Ciossani, Giuseppe
Overlack, Katharina
Voss, Beate
Wohlgemuth, Sabine
Petrovic, Arsen
Wu, Yaowen
Selenko, Philipp
Musacchio, Andrea
Maffini, Stefano
Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology
title Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology
title_full Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology
title_fullStr Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology
title_full_unstemmed Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology
title_short Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology
title_sort electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology
topic Biochemistry and Chemical Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656429/
https://www.ncbi.nlm.nih.gov/pubmed/31310234
http://dx.doi.org/10.7554/eLife.48287
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