Improved split fluorescent proteins for endogenous protein labeling

Siyu Feng, Sayaka Sekine, Veronica Pessino, Han Li, Manuel D. Leonetti and Bo Huang ()
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Siyu Feng: The UC Berkeley-UCSF Graduate Program in Bioengineering
Sayaka Sekine: University of California in San Francisco
Veronica Pessino: University of California, San Francisco
Han Li: University of California, San Francisco
Manuel D. Leonetti: University of California, San Francisco
Bo Huang: University of California in San Francisco

Nature Communications, 2017, vol. 8, issue 1, 1-11

Abstract: 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.

Date: 2017
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DOI: 10.1038/s41467-017-00494-8

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