Intein-mediated reporter gene assay for detecting protein-protein interactions in living mammalian cells.
For nondestructive analysis of chemical processes in living mammalian cells, here we developed a new reporter gene assay for detecting cytosolic protein-protein interactions based on protein splicing of transcription factors with DnaE inteins. The protein splicing induces connection of a DNA-binding protein (modified LexA; mLexA) with a transcription activation domain of a herpes simplex virus protein (VP16AD). We thereby circumvented the limitation of earlier methods for monitoring protein-protein interactions, including the two-hybrid systems, protein complementation systems (PCS), and protein reconstitution systems, and rather combined their advantages. To test the applicability of this method, we monitored epidermal growth factor (EGF)-induced interactions on cell membranes of a known partner, an oncogenic product Ras and its target Raf-1. Ras was connected with N-terminal DnaE and mLexA, while Raf-1 was connected with C-terminal DnaE and VP16AD. Upon stimulation with EGF, the interaction between Ras and Raf-1 triggered folding of the DnaEs, thereby inducing protein splicing to form mLexA-VP16AD fusion protein, and transcription of a reporter gene, firefly luciferase. The extent of Ras-Raf-1 interaction was quantified by measuring the luciferase activity. The interaction was not able to be monitored by two-hybrid systems nor by PCS of split firefly luciferases; however, by using the protein splicing elements and the reporter gene, we obtained the bioluminescence signals sufficient for evaluation of the interactions close to cell membranes.
Kanno
Akira
A
Department of Chemistry, School of Science, The University of Tokyo, Hongo Bunkyo-ku, Tokyo 113-0033, Japan.
Ozawa
Takeaki
T
Umezawa
Yoshio
Y
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017930:Genes, Reporter; D011905:Genes, ras; D047668:Inteins / Q000235:genetics*; D049409:Luciferases, Firefly / Q000235:genetics; D019154:Protein Splicing; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics
D011993:Recombinant Fusion Proteins; D049409:Luciferases, Firefly; D019908:Proto-Oncogene Proteins c-raf