Improved split fluorescent proteins for endogenous protein labeling.

Area of Science:

• Biochemistry
• Molecular Biology
• Microscopy

Background:

• Self-complementing split fluorescent proteins (FPs) are vital tools for protein labeling, subcellular localization, and cell-cell contact detection.
• Existing split FP systems have limitations in signal-to-background ratios and color diversity.

Purpose of the Study:

• To develop a screening strategy for engineering novel self-complementing split FPs.
• To generate improved split FP variants for enhanced cellular imaging applications.

Main Methods:

• Developed a screening strategy for direct engineering of self-complementing split FPs.
• Generated yellow-green split-mNeonGreen2 and red split-sfCherry2 variants.
• Engineered a photoactivatable variant of split-sfCherry2 for super-resolution microscopy.
• Demonstrated dual-color endogenous protein tagging using split sfCherry2 and GFP.

Main Results:

• Created split-mNeonGreen2 (1-10/11) with an improved signal-to-background ratio compared to split GFP.
• Developed a 10-fold brighter red split-sfCherry2 (1-10/11) for multicolor imaging.
• Engineered a photoactivatable split-sfCherry2 variant suitable for super-resolution microscopy.
• Successfully performed dual-color tagging of endogenous proteins, revealing reduced abundance of Sec61B in ER tubules.

Conclusions:

• The new split FPs offer enhanced capabilities for multicolor imaging of endogenous protein interaction networks.
• These FPs provide versatile tools for protein labeling, localization, and advanced microscopy techniques.
• The developed variants hold potential for orthogonal biochemical isolation of native protein complexes.