Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing.
We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp. PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin served to monitor the interaction between IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity. This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system.
Ozawa
T
T
Department of Chemistry, School of Science, The University of Tokyo, Japan.
Kaihara
A
A
Sato
M
M
Tachihara
K
K
Umezawa
Y
Y
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D000458:Cyanobacteria / Q000737:chemistry; D007328:Insulin / Q000737:chemistry; D008156:Luciferases / Q000737:chemistry*; D055095:Optics and Photonics; D011485:Protein Binding; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D007328:Insulin; D011993:Recombinant Fusion Proteins; D008156:Luciferases