Directed evolution of APEX2 for electron microscopy and proximity labeling
Directed evolution of APEX2 for electron microscopy and proximity labeling
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DOI：
10.1038/nmeth.3179
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发表时间：
2015-01-01
期刊：
影响因子：
48
通讯作者：
Ting, Alice Y.
中科院分区：
文献类型：
--
作者：
Lam, Stephanie S.;Martell, Jeffrey D.;Ting, Alice Y.
APEX是一种工程化的过氧化物酶，作为电子显微镜标签和活细胞蛋白质组学的混杂标记酶。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。APEX 2在细胞中更活跃，使得能够使用电子显微镜来解析钙摄取调节蛋白MICU 1的亚线粒体定位。APEX 2还允许内源性线粒体和内质网膜蛋白的上级富集。
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.