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:
Article
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
Descripción
Sumario: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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