Figure 4
From: A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association
Characterization of protein-protein interactions using coiled-coil heterodimerization.
(a) Expression vectors for tripartite split-GFP interaction assays in E. coli: bicistronic pTET tetracycline inducible vector harboring GFP10 and GFP11 tags with cloning sites for test proteins; IPTG inducible pET vector (T7 promoter) for stable expression of GFP1–9 OPT. (b) Colony fluorescence on plates after Antet and IPTG co-induction driving expression of GFP10-K1/E1-GFP11 pair (K/E) (top) and GFP10-E1/E1-GFP11 (E1/E1) (bottom) with GFP1–9 detection fragment. (c) In vitro quantification of K1/E1 or E1/E1 interaction with increasing amount of E-GFP11 (0.4 nM up to 800 nM). Fluorescence values 1 h after initiating complementation with GFP1–9. (d) Initial velocity rates of complementation reactions with various concentrations of GFP1–9 detection reagent (0.5 μM up to 8 μM) in GFP10-K1/E1-GFP11 assay (1 μM of each tagged species).
