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APEX2 的定向进化用于电子显微镜和邻近标记。

Directed evolution of APEX2 for electron microscopy and proximity labeling.

机构信息

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.

出版信息

Nat Methods. 2015 Jan;12(1):51-4. doi: 10.1038/nmeth.3179. Epub 2014 Nov 24.

DOI:10.1038/nmeth.3179
PMID:25419960
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4296904/
Abstract

APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.

摘要

APEX 是一种工程过氧化物酶，可作为电子显微镜标记物和用于活细胞蛋白质组学的广谱标记酶。由于有限的灵敏度排除了需要低 APEX 表达的应用，我们使用酵母展示进化来提高其催化效率。APEX2 在细胞中的活性要高得多，这使得可以使用电子显微镜来解析钙摄取调节蛋白 MICU1 的亚线粒体定位。APEX2 还可以更好地富集内源性线粒体和内质网膜蛋白。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45f6/4296904/7e52325c6af0/nihms639105f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45f6/4296904/1f062d989caa/nihms639105f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45f6/4296904/0e9af430da66/nihms639105f2a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45f6/4296904/7e52325c6af0/nihms639105f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45f6/4296904/1f062d989caa/nihms639105f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45f6/4296904/0e9af430da66/nihms639105f2a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/45f6/4296904/7e52325c6af0/nihms639105f3.jpg

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Directed evolution of APEX2 for electron microscopy and proximity labeling.

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[7]
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本文引用的文献

[1]
Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging.

Mol Cell. 2014-7-4

[2]
MICU1 and MICU2 play nonredundant roles in the regulation of the mitochondrial calcium uniporter.

EMBO Rep. 2014-2-6

[3]
MICU1 motifs define mitochondrial calcium uniporter binding and activity.

Cell Rep. 2013-12-12

[4]
MICU1 controls both the threshold and cooperative activation of the mitochondrial Ca²⁺ uniporter.

Cell Metab. 2013-6-4

[5]
Photooxidation-guided ultrastructural identification and analysis of cells in neuronal tissue labeled with green fluorescent protein.

PLoS One. 2013-5-31

[6]
MICU2, a paralog of MICU1, resides within the mitochondrial uniporter complex to regulate calcium handling.

PLoS One. 2013-2-7

[7]
Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.

Science. 2013-1-31

[8]
MICU1 is an essential gatekeeper for MCU-mediated mitochondrial Ca(2+) uptake that regulates cell survival.

Cell. 2012-10-26

[9]
Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy.

Nat Biotechnol. 2012-10-21

[10]
A transcription activator-like effector toolbox for genome engineering.

Nat Protoc. 2012-1-5

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