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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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
2014
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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. |
format | Online Article Text |
id | pubmed-4296904 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
record_format | MEDLINE/PubMed |
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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