Detecting protein-protein interactions with GFP-fragment reassembly

The detection of protein-protein interactions in vivo is of critical importance to our understanding of biological processes. The classic library approach has been to use the yeast two-hybrid screen, where an interaction between known bait and unknown prey proteins leads to restoration of transcription factor activity(1). However, its use is limited by host organism and nuclear localization requirements, and a tendency to detect indirect interactions (false positives). Bacterial two-hybrid screens have eliminated localization requirements and simplified many technical aspects of the procedure'. An innovative approach has been the reassembly of protein fragments, which then directly report interactions. A suitable reporter protein is dissected at the genetic level, and the fragments are fused to bait and prey, which are then coexpressed Bait and prey interaction brings the reporter fragments together, facilitating reassembly of the active reporter protein, giving a direct readout of the association. This method has been demonstrated for dihydrofolate reductase3,4, ubiquitin5 and the green fluorescent protein6 (GFP) from Aequorea victoria. We recently described improvements to the original screen based on the reassembly of the GFP enhancedstability mutant sg(100) in Escherichia colil. Our system, presented in the protocol that follows, consists of two plasmid vectors for the independent expression of fusions with N- and C-terminal fragments of GFP, and allows for simple visual detection of protein-protein interactions with a K-D as weak as 1 mM.