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Directed evolution of APEX2 for electron microscopy and proteomics

APEX is an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. Because the limited sensitivity of APEX precludes applications requiring low APEX expression, we used yeast display evolution to improve its catalytic ef...

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Detalles Bibliográficos
Autores principales: Lam, Stephanie S., Martell, Jeffrey D., Kamer, Kimberli J., Deerinck, Thomas J., Ellisman, Mark H., Mootha, Vamsi K., Ting, Alice Y.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4296904/
https://www.ncbi.nlm.nih.gov/pubmed/25419960
http://dx.doi.org/10.1038/nmeth.3179
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author Lam, Stephanie S.
Martell, Jeffrey D.
Kamer, Kimberli J.
Deerinck, Thomas J.
Ellisman, Mark H.
Mootha, Vamsi K.
Ting, Alice Y.
author_facet Lam, Stephanie S.
Martell, Jeffrey D.
Kamer, Kimberli J.
Deerinck, Thomas J.
Ellisman, Mark H.
Mootha, Vamsi K.
Ting, Alice Y.
author_sort Lam, Stephanie S.
collection PubMed
description APEX is an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. Because the limited sensitivity of APEX precludes applications requiring low APEX expression, we used yeast display evolution to improve its catalytic efficiency. Our evolved APEX2 is far more active in cells, enabling the superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins and the use of electron microscopy to resolve the sub-mitochondrial localization of calcium uptake regulatory protein MICU1.
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spelling pubmed-42969042015-07-01 Directed evolution of APEX2 for electron microscopy and proteomics Lam, Stephanie S. Martell, Jeffrey D. Kamer, Kimberli J. Deerinck, Thomas J. Ellisman, Mark H. Mootha, Vamsi K. Ting, Alice Y. Nat Methods Article APEX is an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. Because the limited sensitivity of APEX precludes applications requiring low APEX expression, we used yeast display evolution to improve its catalytic efficiency. Our evolved APEX2 is far more active in cells, enabling the superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins and the use of electron microscopy to resolve the sub-mitochondrial localization of calcium uptake regulatory protein MICU1. 2014-11-24 2015-01 /pmc/articles/PMC4296904/ /pubmed/25419960 http://dx.doi.org/10.1038/nmeth.3179 Text en http://www.nature.com/authors/editorial_policies/license.html#terms Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms
spellingShingle Article
Lam, Stephanie S.
Martell, Jeffrey D.
Kamer, Kimberli J.
Deerinck, Thomas J.
Ellisman, Mark H.
Mootha, Vamsi K.
Ting, Alice Y.
Directed evolution of APEX2 for electron microscopy and proteomics
title Directed evolution of APEX2 for electron microscopy and proteomics
title_full Directed evolution of APEX2 for electron microscopy and proteomics
title_fullStr Directed evolution of APEX2 for electron microscopy and proteomics
title_full_unstemmed Directed evolution of APEX2 for electron microscopy and proteomics
title_short Directed evolution of APEX2 for electron microscopy and proteomics
title_sort directed evolution of apex2 for electron microscopy and proteomics
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4296904/
https://www.ncbi.nlm.nih.gov/pubmed/25419960
http://dx.doi.org/10.1038/nmeth.3179
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