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Nat Commun. 2017 Aug 29;8(1):370. doi: 10.1038/s41467-017-00494-8.

Improved split fluorescent proteins for endogenous protein labeling.

Author information

1
The UC Berkeley-UCSF Graduate Program in Bioengineering, San Francisco, CA, 94143, USA.
2
Department of Pharmaceutical Chemistry, University of California in San Francisco, San Francisco, CA, 94143, USA.
3
Graduate Program of Biophysics, University of California, San Francisco, San Francisco, CA, 94143, USA.
4
Department of Cellular and Molecular Pharmacology, and Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, 94143, USA.
5
Department of Pharmaceutical Chemistry, University of California in San Francisco, San Francisco, CA, 94143, USA. bo.huang@ucsf.edu.
6
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, 94143, USA. bo.huang@ucsf.edu.
7
Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA. bo.huang@ucsf.edu.

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.Split fluorescent proteins (FPs) have been widely used to visualise proteins in cells. Here the authors develop a screen for engineering new split FPs, and report a yellow-green split-mNeonGreen2 with reduced background, a red split-sfCherry2 for multicolour labeling, and its photoactivatable variant for super-resolution use.

PMID:
28851864
PMCID:
PMC5575300
DOI:
10.1038/s41467-017-00494-8
[Indexed for MEDLINE]
Free PMC Article

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