Bioluminescent Imaging of MAPK Function with Intein-Mediated Reporter Gene Assay

For nondestructive analysis of chemical processes in living mammalian cells, here we show a new reporter gene assay for detecting Ras–Raf-1 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). Ras is connected with N-terminal DnaE and mLexA, while Raf-1 is connected with C-terminal DnaE and VP16AD. Upon stimulation with EGF, the interaction between Ras and Raf-1 triggers 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 is quantified by measuring the luciferase activity. By using the protein-splicing elements and the reporter gene, the Ras–Raf-1 interaction close to cell membranes can be evaluated.