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.
Highlights
We report a screening strategy for the direct engineering of self-complementing split fluorescent proteins (FPs). Using this strategy, we have generated a yellow–green-colored mNeonGreen21–10/11 (mNG2) that has an improved ratio of complemented signal to the background of FP1–10-expressing cells as compared to GFP1–10/11, as well as a red-colored sfCherry21–10/11 that is about a
Background from untagged cells293T WT SFFV_GFP1–10 PGK_GFP1–10 SFFV_mNG21–10Log% of max % of max % of max c LMNAGFP11 (PGK_GFP1–10)mNG211 (SFFV_mNG21–10) RAB11A 1201234 Log 10 μm d e% of max Relative fluorescence intensity SPTLC1
Inspired by assays previously used to optimize a protease reporter[9], we devised a general strategy for the engineering of selfcomplementing split fluorescent proteins (FPs)
Summary
We report a screening strategy for the direct engineering of self-complementing split FPs. Using this strategy, we have generated a yellow–green-colored mNeonGreen21–10/11 (mNG2) that has an improved ratio of complemented signal to the background of FP1–10-expressing cells as compared to GFP1–10/11, as well as a red-colored sfCherry21–10/11 that is about a With 11:1–10 transfected DNA ratio increased from 1:1 to 1:3 and 1:5, the normalized whole cell fluorescence by flow cytometry from mNG211 is approximately 50–60% of that from full length mNG2 (Fig. 2e).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
