A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association

Abstract

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments

“…Lack of detection of the reconstituted GFP signal in this study could be due to a strong background signal at the infection site that masked the low complemented GFP signal (Tanaka et al, 2015). Since the original sfGFP1-10 (Cabantous et al, 2005) was used in the study, GFP complementation might have occurred less efficiently due to the aggregation of sfGFP1-10 ( Cabantous et al, 2013). Here, we generated a plant expression system using sfGFP1-10 OPT , which is optimized to overcome the protein aggregation issue (Cabantous et al, 2013).…”

“…Since the original sfGFP1-10 (Cabantous et al, 2005) was used in the study, GFP complementation might have occurred less efficiently due to the aggregation of sfGFP1-10 ( Cabantous et al, 2013). Here, we generated a plant expression system using sfGFP1-10 OPT , which is optimized to overcome the protein aggregation issue (Cabantous et al, 2013). Our vector set with the AvrRpm1 effector signal peptide should facilitate localization studies of fungal and other plant pathogen effectors.…”

“…Once GFP1-10 and GFP11 exist in close proximity, the two fragments assemble a barrel structure and then emit fluorescence. The original split GFP system (Ghosh et al, 2000) has been further engineered to improve protein folding kinetics and solubility to enhance fluorescence intensity (Cabantous et al, 2005;Pédelacq et al, 2006;Cabantous et al, 2013). For better protein folding efficiency, super-folder GFP (sfGFP) was engineered for split GFP system (Pédelacq et al, 2006).…”

“…However, the solubility of the sfGFP1-10 fragment is poor, resulting in lower fluorescence intensity (Cabantous et al, 2005). Therefore, a variant of sfGFP1-10 was engineered called GFP1-10 OPT that significantly improves solubility and fluorescence intensity (Cabantous et al, 2005;Cabantous et al, 2013). In addition, recently, single amino acid mutants of sfGFP1-10 OPT were generated that show emission spectra shifted to yellow or cyan color .…”

A FACS-based screening strategy to assess sequence-specific RNA-binding of Pumilio protein variants in E. coli

“…TetFC uses two variants of the RNA-binding protein Pumilio and a three-partite split-GFP ( Figure 1A) (Cabantous et al, 2013;Kellermann et al, 2013). Each of the two Pumilio proteins is tagged with one of the β-strands of GFP (S10 and S11, respectively).…”