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Bright split red fluorescent proteins with enhanced complementation efficiency for the tagging of endogenous proteins and visualization of synapses

Siyu Feng, Aruna Varshney, Doris Coto Villa, Cyrus Modavi, John Kohler, Fatima Farah, Nebat Ali, Joachim Dieter Mueller, Miri VanHoven, Bo Huang
doi: https://doi.org/10.1101/454041
Siyu Feng
The UC Berkeley-UCSF Graduate Program in Bioengineering, San Francisco, CA 94143 USA;
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Aruna Varshney
Department of Biological Sciences, San Jose State University, San Jose, CA 95192 USA;
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Doris Coto Villa
Department of Biological Sciences, San Jose State University, San Jose, CA 95192 USA;
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Cyrus Modavi
Department of Bioengineering and Therapeutic Sciences, University of California in San Francisco, San Francisco, CA 94143, USA;
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John Kohler
School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA;
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Fatima Farah
Department of Biological Sciences, San Jose State University, San Jose, CA 95192 USA;
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Nebat Ali
Department of Biological Sciences, San Jose State University, San Jose, CA 95192 USA;
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Joachim Dieter Mueller
School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA;
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Miri VanHoven
Department of Biological Sciences, San Jose State University, San Jose, CA 95192 USA;
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Bo Huang
Department of Pharmaceutical Chemistry, University of California in San Francisco, San Francisco, CA 94143, USA;Department Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143, USA;Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.
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  • For correspondence: bo.huang@ucsf.edu
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Abstract

Self-associating split fluorescent proteins (FPs) have been widely used for labeling proteins, scaffolding protein assembly and detecting cell-cell contacts. Newly developed self-associating split FPs, however, have suffered from suboptimal fluorescence signal. Here, by investigating the complementation process, we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution. The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness, facilitating the tagging of endogenous proteins by gene editing. Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) for multiplexed visualization of neuronal synapses in living animals, demonstrating its broad applications.

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Posted October 25, 2018.
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Bright split red fluorescent proteins with enhanced complementation efficiency for the tagging of endogenous proteins and visualization of synapses
Siyu Feng, Aruna Varshney, Doris Coto Villa, Cyrus Modavi, John Kohler, Fatima Farah, Nebat Ali, Joachim Dieter Mueller, Miri VanHoven, Bo Huang
bioRxiv 454041; doi: https://doi.org/10.1101/454041
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Bright split red fluorescent proteins with enhanced complementation efficiency for the tagging of endogenous proteins and visualization of synapses
Siyu Feng, Aruna Varshney, Doris Coto Villa, Cyrus Modavi, John Kohler, Fatima Farah, Nebat Ali, Joachim Dieter Mueller, Miri VanHoven, Bo Huang
bioRxiv 454041; doi: https://doi.org/10.1101/454041

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