Open AccessImproved split fluorescent proteins for endogenous protein labeling
Author(s) -
Siyu Feng,
Sayaka Sekine,
Veronica Pessino,
Han Li,
Manuel D. Leonetti,
Bo Huang
Publication year - 2017
Publication title -
nature communications
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.559
H-Index - 365
ISSN - 2041-1723
DOI - 10.1038/s41467-017-00494-8
Subject(s) - green fluorescent protein , endoplasmic reticulum , endogeny , protein subcellular localization prediction , protein sorting signals , fluorescence , microbiology and biotechnology , superresolution , fluorescence microscope , chemistry , biology , computational biology , signal peptide , biochemistry , computer science , peptide sequence , gene , artificial intelligence , physics , image (mathematics) , quantum mechanics
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-mNeonGreen2 1–10/11 that improves the ratio of complemented signal to the background of FP 1–10 -expressing cells compared to the commonly used split GFP 1–10/11 ; as well as a 10-fold brighter red-colored split-sfCherry2 1–10/11 . 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 sfCherry2 11 and GFP 11 , 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.