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Improved Split Fluorescent Proteins for Endogenous Protein Labeling

Siyu Feng, Sayaka Sekine, Veronica Pessino, Han Li, Manuel D. Leonetti, Bo Huang
doi: https://doi.org/10.1101/137059
Siyu Feng
1The UC Berkeley-UCSF Graduate Program in Bioengineering, San Francisco, CA 94143 USA;
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Sayaka Sekine
2Department of Pharmaceutical Chemistry, University of California in San Francisco, San Francisco, CA 94143, USA;
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Veronica Pessino
3Graduate Program of Biophysics, University of California, San Francisco, San Francisco, CA 94143, USA;
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Han Li
4Department of Cellular and Molecular Pharmacology, and Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94143, USA;
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Manuel D. Leonetti
4Department of Cellular and Molecular Pharmacology, and Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94143, USA;
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Bo Huang
2Department of Pharmaceutical Chemistry, University of California in San Francisco, San Francisco, CA 94143, USA;
5Department Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143, USA.
6Chan Zuckerberg Biohub, San Francisco, CA 94158, USA
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  • For correspondence: bo.huang@ucsf.edu
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ABSTRACT

Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen21-10/11 that improves the ratio of complemented signal to the background of FP1-10-expressing cells compared to the commonly used split-GFP1-10/11, as well as a 10-fold brighter red-colored split-sfCherry21-10/11. Based on split-sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted July 26, 2017.
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Improved Split Fluorescent Proteins for Endogenous Protein Labeling
Siyu Feng, Sayaka Sekine, Veronica Pessino, Han Li, Manuel D. Leonetti, Bo Huang
bioRxiv 137059; doi: https://doi.org/10.1101/137059
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Improved Split Fluorescent Proteins for Endogenous Protein Labeling
Siyu Feng, Sayaka Sekine, Veronica Pessino, Han Li, Manuel D. Leonetti, Bo Huang
bioRxiv 137059; doi: https://doi.org/10.1101/137059

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