RT Journal Article SR Electronic T1 Improved Split Fluorescent Proteins for Endogenous Protein Labeling JF bioRxiv FD Cold Spring Harbor Laboratory SP 137059 DO 10.1101/137059 A1 Siyu Feng A1 Sayaka Sekine A1 Veronica Pessino A1 Han Li A1 Manuel D. Leonetti A1 Bo Huang YR 2017 UL http://biorxiv.org/content/early/2017/05/12/137059.abstract AB 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 generated a yellow-green colored split mNeonGreen21-10/11 with greatly reduced background from the uncomplemented 1-10 fragment, as compared to the commonly used split GFP1-10/11; as well as a 10 times 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.