期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 117, 期 52, 页码 33186-33196出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2006521117
关键词
protein engineering; proteases neuroscience
资金
- Stanford University
- Chan Zuckerberg Biohub
- Beckman Technology Development Seed Grant
- NIH [R01 MH119353, F32 NS106764, R01 NS94668]
- EMBO long-term postdoctoral fellowship [ALTF 1022-2015]
Molecular integrators, in contrast to real-time indicators, convert transient cellular events into stable signals that can be exploited for imaging, selection, molecular characterization, or cellular manipulation. Many integrators, however, are designed as complex multicomponent circuits that have limited robustness, especially at high, low, or nonstoichiometric protein expression levels. Here, we report a simplified design of the calcium and light dual integrator FLARE. Single-chain FLARE (scFLARE) is a single polypeptide chain that incorporates a transcription factor, a LOV domain-caged protease cleavage site, and a calcium-activated TEV protease that we designed through structure-guided mutagenesis and screening. We show that scFLARE has greater dynamic range and robustness than first-generation FLARE and can be used in culture as well as in vivo to record patterns of neuronal activation with 10-min temporal resolution.
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