Improved split fluorescent proteins for endogenous protein labeling

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.

LMO7 deficiency reveals the significance of the cuticular plate for hearing function

“…This strategy allows tagging and visualization of the protein of interest based on the localization of reconstituted GFP fluorescence. Split fluorescent protein has been widely used for protein quantification, visualization of protein cellular localization, as well as single-molecule imaging and protein-protein interaction [36][37][38][39][40][41][42] . Using CRISPR/Cas genome editing, we knocked-in the GFP11-coding sequence (48 nucleotides) into the C-terminus of mouse Lmo7, immediately prior to the stop codon (resulting in the Lmo7-GFP11 KI mice) ( Fig.…”

Experimental observation of node-line-like surface states in LaBi

“…Our first-principles calculations reveal that these "nodal lines" have a tiny gap, which is beyond typical experimental resolution. These results may provide important information to understand the extraordinary physical properties of LaBi, such as the extremely large magnetoresistance and resistivity plateau.Topological semimetals represent a novel class of quantum materials whose conduction and valence bands touch at discrete points or extended lines [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. Until now, three types of topological semimetals have been discovered: Dirac, Weyl and nodal line.…”

“…While the Dirac cones or nodal lines discovered in topological semimetals typically derive from the bulk bands, an interesting question is that whether they can exist as surface states of three-dimensional crystals. The nodal line surface states could manifest extraordinary properties that are distinct from other two-dimensional Dirac materials [18][19][20]. To date, the surface Dirac cones have already been extensively studied in topological insulators, such as the Bi 2 Se 3 family materials [21][22][23], while theoretical and experimental studies on the surface nodal lines are still rare [12].…”

Targeted Cleavage and Polyadenylation of RNA by CRISPR-Cas13

“…2). sfGFP forms a beta barrel comprised of 11 antiparallel beta strands, which can tolerate sequence insertions between the 10 th and 11 th beta strands but loses fluorescence if the 11 th beta strand is removed [26][27][28] . We generated an sfGFP reporter construct with the coding sequence for the 11 th beta strand embedded within a prototypical mammalian intron (second intron of the rabbit beta globin gene) (sfGFPapa) (Fig.…”