US 12,247,244 B2
Membrane protein activity measurement method
Yuichi Ikeda, Tokyo (JP); and Hidetoshi Kumagai, Tokyo (JP)
Assigned to THE UNIVERSITY OF TOKYO, Tokyo (JP)
Appl. No. 17/263,410
Filed by THE UNIVERSITY OF TOKYO, Tokyo (JP)
PCT Filed Jul. 26, 2019, PCT No. PCT/JP2019/029424
§ 371(c)(1), (2) Date May 3, 2021,
PCT Pub. No. WO2020/026979, PCT Pub. Date Feb. 6, 2020.
Claims priority of application No. 2018-144171 (JP), filed on Jul. 31, 2018.
Prior Publication US 2021/0310039 A1, Oct. 7, 2021
Int. Cl. C12Q 1/06 (2006.01); C12N 15/10 (2006.01); C12N 15/62 (2006.01); C12N 15/85 (2006.01); C12Q 1/66 (2006.01)
CPC C12Q 1/06 (2013.01) [C12N 15/1086 (2013.01); C12N 15/62 (2013.01); C12N 15/85 (2013.01); C12Q 1/66 (2013.01)] 5 Claims
OG exemplary drawing
 
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.