Cargando…
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...
Autores principales: | , , , , , , , , , , , , , |
---|---|
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 |
_version_ | 1783438641832394752 |
---|---|
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. |
format | Online Article Text |
id | pubmed-6656429 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT alexamal electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT pianovalentina electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT polleysoumitra electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT stuivermarchel electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT vossstephanie electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT ciossanigiuseppe electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT overlackkatharina electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT vossbeate electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT wohlgemuthsabine electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT petrovicarsen electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT wuyaowen electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT selenkophilipp electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT musacchioandrea electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology AT maffinistefano electroporatedrecombinantproteinsastoolsforinvivofunctionalcomplementationimagingandchemicalbiology |