US 12,459,985 B2
G-protein-coupled receptor internal sensors
Lin Tian, Davis, CA (US); Tommaso Patriarchi, Davis, CA (US); and Ruqiang Liang, Davis, CA (US)
Assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, Oakland, CA (US)
Appl. No. 16/463,313
Filed by THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, Oakland, CA (US)
PCT Filed Nov. 22, 2017, PCT No. PCT/US2017/062993
§ 371(c)(1), (2) Date May 22, 2019,
PCT Pub. No. WO2018/098262, PCT Pub. Date May 31, 2018.
Claims priority of provisional application 62/513,991, filed on Jun. 1, 2017.
Claims priority of provisional application 62/426,173, filed on Nov. 23, 2016.
Prior Publication US 2021/0101960 A1, Apr. 8, 2021
Int. Cl. C07K 14/71 (2006.01); A61K 49/00 (2006.01); C07K 14/72 (2006.01); G01N 33/533 (2006.01); G01N 33/542 (2006.01); G01N 33/74 (2006.01)
CPC C07K 14/723 (2013.01) [A61K 49/0008 (2013.01); A61K 49/0056 (2013.01); G01N 33/542 (2013.01); G01N 33/74 (2013.01); C07K 2319/60 (2013.01); G01N 2333/726 (2013.01)] 14 Claims
OG exemplary drawing
 
1. A class A type G protein-coupled receptor (GPCR) comprising a fluorescent sensor, the sensor comprising the following polypeptide structure:
L1-cpFP-L2, wherein:
(1) L1 comprises a peptide linker LSSLI (SEQ ID NO:73);
(2) cpFP comprises a circularly permuted beta barrel fluorescent protein, wherein the circularly permuted N-terminus is positioned within beta strand seven of a non-permuted beta barrel fluorescent protein; and
(3) L2 comprises a peptide linker NHDQL (SEQ ID NO:74), and
wherein the GPC rises SEQ ID NO. 165;
wherein the circularly permuted fluorescent protein is from a fluorescent protein having at least 90% sequence identity to a non-permuted fluorescent protein selected from the group consisting of SEQ ID NO's: 1-14 or wherein the circularly permuted fluorescent protein has at least 90% sequence identity to a circularly permuted fluorescent protein selected from the group consisting of SEQ ID NOs: 15-18;
wherein the insertion site for the sensor is located in the third intracellular loop of SEQ ID NO: 165;
wherein the insertion site in the GPCR for the sensor is located after amino acid residue 228 of SEQ ID NO: 165; and
wherein the insertion site for the sensor is located between Q229 and L230, is located between S246 and Q247, replaces the amino acid residues between R228 and K267, or replaces the amino acid residues between L230 and F264.