A Split Enhanced Green Fluorescent Protein-Based Reporter in Yeast Two-Hybrid System

Abstract

We have developed a novel reporter system involving a yeast two-hybrid assay, which utilizes the reconstitution of the split EGFP reporter in order to characterize the relevant protein-protein interactions. To our knowledge, this study represents the first application of the split EGFP system as a read-out in a yeast two-hybrid assay. In comparison with the existing two-hybrid system, the bait and prey vectors were improved with regard to the reporter and the replication control element. As a result, the reconstituted EGFP has been observed to evidence a restored fluorescence upon protein-protein interactions in yeast, thereby allowing for the characterization of its interactor. The use of a split EGFP reporter has some salient advantages. Firstly, no substrates are required for the production of fluorescence. Secondly, low copy number plasmids may help to solve the protein toxicity problem, via the reduction of expression. Thirdly, this technique may prove useful in overcoming the autoactivation problem, due to the fact that the read-out of the yeast two-hybrid system is transcription-independent. Collectively, our results showed that the split EGFP reporter system might potentially be applied in yeast two-hybrid assays for the high-throughput screening of protein-protein interactions, with a simple and direct fluorescent read-out.

Detection and localisation of protein–protein interactions in Saccharomyces cerevisiae using a split-GFP method

“…Recently, two methods for implementing BiFC in S. cerevisiae have been described. The first used plasmids carrying hapto-EGFP genes into which genes encoding the proteins of interest were introduced prior to transformation into the yeast cells (Park et al, 2007). A plasmid-based BiFC assay was used to monitor the cell-cycle dependent association and localisation of Hof1p and Grr1p (Blondel et al, 2005).…”

“…The ability of certain non-fluorescent fragments of GFP (referred to here as hapto-GFPs) to re-assemble in vivo and regain fluorescence provides the basis for these methods. BiFC assays have been applied to interactions occurring in a range of organisms including bacteria (Magliery et al, 2005), fungi (Hoff and Kuck 2005;Park et al, 2007;Sung and Huh 2007), plants (Bracha-Drori et al, 2004;Citovsky et al, 2006) and mammalian cells (Hu et al, 2002;Latz et al, 2007). They work in a similar way to PCAs, with the additional advantage that they require no additional cofactors or enzymatic substrates due to the autocatalytic formation of the intrinsic fluorophore of GFP and its variants (Chalfie, 1995;Cody et al, 1993;Morell et al, 2007;Yang et al, 1996).…”

Bimolecular Fluorescence Complementation (BiFC) Analysis as a Probe of Protein Interactions in Living Cells

“…Many interactions have been visualized in Escherichia coli (6,33,69,75) Agrobacterium tumefaciens (15,113) and Bacillus subtilis (77,105). Among fungi, BiFC analysis has been extensively used in Saccharomyces cerevisiae (bakers yeast) (8,55,84), and also in Acremonium chrysogenum (44), Aspergillus nidulans (9) and Magnaporthe grisea (127). Among higher eukaryotic organisms, BiFC analysis has been used to visualized numerous interactions in many plant species (1,5,11,12,14 ,118,119,124,125).…”

Two-hybrid-based systems: Powerful tools for investigation of membrane traffic machineries

“…No need of substrates or cofactors; sensitivity, flexibility and detection of weak interactions (Park et al, 2007;Blakeley et al, 2012).…”