Cell chemical biology · 2026
Directed evolution of APOX for proximity labeling using phenols with high redox potentials.
СтатьяНазвание и аннотация: Перевод в очереди
Обработка статьи · этапы, ошибки и управление
Загрузка состояния…
Ошибки и ограничения
История · было → стало
Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.
Fang Sifei, Acevedo Leslie D, Solivais Alexander J, Zhou Xu, Patil Swapnali S, Delfosse Elliot S, Yin Hang, Ziolkowski Zachary B, Frey Brian L, Chapman Edwin R, Smith Lloyd M, Martell Jeffrey D
Аннотация
Proximity labeling (PL) can identify endogenous proteins in specific subcellular regions. APEX2 (enhanced ascorbate peroxidase 2) enables PL with high temporal resolution by oxidizing biotin phenol (BP) into a radical that promiscuously tags nearby proteins, followed by the isolation and identification of the tagged biomolecules. However, the BP radical has a relatively large diffusion radius, limiting spatial resolution. Replacing the phenol in BP with nitrophenol (NP) could shorten the labeling radius by shortening the radical lifetime, but existing PL heme peroxidases cannot efficiently oxidize substrates with high redox potentials. We report the ultrahigh-throughput directed evolution of APOX (APEX with high redOX potential), a quadruple mutant of APEX2 with a higher reduction potential. Using APOX with a membrane-permeable alkyne-NP probe, we demonstrate PL in living mammalian cells, including proteomic mapping with excellent subcellular compartment specificity.