Membrane protein activity measurement method
The present invention provides a comprehensive assay capable of comprehensively measuring interactive factors for a variety of different types of proteins. In the present invention, provided is a system for detecting activation of a protein by integrating, with mediation of a plurality of factors, changes in the transcriptional regulatory region of a gene whose expression is changed by activation of the protein. The stimulation response ratio of transcription activity before and after stimulation by ligands possessed by individual transcriptional regulatory regions is synergistically integrated, so that a highly sensitive reporter assay system can be constructed.
1. A combination of constructs for investigating the action of a test compound on a protein responsible for signal transduction,
wherein the combination of constructs comprises n sequential constructs for a sequential enzyme reaction cascade and one reporter gene construct,
wherein
the n constructs for a sequential enzyme reaction cascade comprises one nucleotide sequence encoding a substrate operably linked to the transcriptional regulatory region of a gene (gene 1) and n−1 nucleotide sequences, wherein the each one of the n−1 nucleotide sequences encodes a different enzyme operably linked to the transcriptional regulatory region of a different gene out of n genes other than gene 1, respectively,
the expressions of n genes, whose transcriptional regulatory regions are used in n sequential constructs for a sequential enzyme reaction cascade, are induced by activation of the protein,
the reporter gene construct is configured to activate the reporter gene by the final product of the sequential enzyme reaction cascade,
n is an integer of 3 or more, and
the combination includes:
(1) a first construct in which the transcriptional regulatory region of a first gene (gene 1) is linked to a nucleotide sequence encoding a substrate(S) for an enzyme reaction;
(2) a second construct in which the transcriptional regulatory region of a second gene (gene 2) is linked to a nucleotide sequence encoding an enzyme (E1) for an enzyme reaction, wherein a product P1 is generated as a result of the enzyme reaction between S and E1
(3) n−2 sequential construct(s): each construct encodes an enzyme, which generates a product by catalyzing the previous product generated by the previous enzyme reaction mediated by the previous enzyme encoded in the previous construct, is operably linked to the transcriptional regulatory regions of a gene out of n genes other than previously used genes, and
(4) a reporter gene construct that is configured so that a reporter gene is activated by a final product of a sequential enzyme reaction cascade, wherein the expressions of n genes, whose transcriptional regulatory regions are used in n constructs for a sequential enzyme reaction cascade, are induced by stimulation of the protein by the test compound.
2. A cell comprising the combination of constructs according to claim 1 .
3. The combination of constructs according to claim 1 , wherein n=3.
4. The cell according to claim 2 , wherein n=3.
5. The cell according to claim 4 , wherein the cell is a HeLa cell.