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公开(公告)号：US20170234870A1
公开(公告)日：2017-08-17
申请号：US15518888
申请日：2015-10-14
IPC分类号： G01N33/566 , G01N21/64 , C07K14/47 , G01N33/542 , C12Q1/66
CPC分类号： G01N33/566 , C07K14/4722 , C07K14/723 , C07K2319/00 , C07K2319/01 , C07K2319/60 , C12N9/0069 , C12Q1/66 , C12Y113/12007 , G01N21/6428 , G01N33/542 , G01N2021/6439 , G01N2333/4719 , G01N2333/726 , G01N2500/02
摘要： Resonance energy transfer (RET)- or protein-fragment complement assay (PCA)-based biosensors useful for assessing the activity of G-proteins are described. These biosensors are based on the competition between the Got subunit and a Gβγ interacting protein (βγ IP) for the binding to the Gβγ dimer. These biosensors comprises (1) a βγ IP and (2) a Gβ or Gγ protein; a GPCR; or a plasma membrane targeting domain, fused to suitable RET or PCA tags. Methods using such biosensors for different applications, including the identification of agents that modulates G-protein activity or for the characterization of GPCR signaling/regulation, such as G-protein preferences and activation profiles of GPCRs, are also described.
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公开(公告)号：US20170313762A1
公开(公告)日：2017-11-02
申请号：US15512267
申请日：2015-09-21
申请人： The Royal Institution for the Advancement of Learning/McGill University , Université de Montréal
发明人： Stéphane Alain Laporte , Yoon Namkung , Michel Bouvier , Christian Le Gouill , Mirelle Hogue , Viktoriya Lukasheva , Hiroyuki Kobayashi , Denis Deblois , Étienne Durette
摘要： Bioluminescence resonance energy transfer (BRET) biosensors for assessing the intracellular localization, internalization and trafficking into cellular compartments of proteins such as receptors, and other biomolecules such as second messengers, are disclosed. These biosensors, which are dependent on the concentration/density of the BRET donor and acceptor in cellular compartments rather that specific protein-protein interactions, use a Renilla GFP/Luc BRET pair, which allows the robust and reproducible monitoring of protein trafficking/localization, with a sensitivity compatible with high-throughput screening (HTS). The use of these biosensors for various applications, including assessing/monitoring protein endocytosis, recycling and intra-cellular trafficking, receptor maturation/rescue by pharmacological chaperones, various endocytosis/exocytosis processes, activation/inhibition, as well as biomolecule concentration/density in different cellular compartments, is also disclosed.
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公开(公告)号：US20200256869A1
公开(公告)日：2020-08-13
申请号：US16755976
申请日：2018-10-15
IPC分类号： G01N33/566 , G01N33/542
摘要： Systems and methods for the monitoring of G protein activation at various cell compartments, such as the plasma membrane and the endosomes, and in a Gα protein subunit family-selective manner are described. These systems and methods also allows the monitoring of G protein-coupled receptor (GPCR)-mediated as well as non-receptor guanine nucleotide exchange factor (GEF)-mediated G protein activation, and are based on the use of the G protein-binding domains of specific effectors of G proteins and cellular compartment markers, tagged with suitable energy (BRET) donors and acceptors.
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4.发明授权公开(公告)号：US10584159B2
公开(公告)日：2020-03-10
申请号：US15512267
申请日：2015-09-21
申请人： The Royal Institution for the Advancement of Learning/McGill University , Université de Montréal
发明人： Stéphane Alain Laporte , Yoon Namkung , Michel Bouvier , Christian Le Gouill , Mireille Hogue , Viktoriya Lukasheva , Hiroyuki Kobayashi , Denis Deblois , Étienne Durette
摘要： Bioluminescence resonance energy transfer (BRET) biosensors for assessing the intracellular localization, internalization and trafficking into cellular compartments of proteins such as receptors, and other biomolecules such as second messengers, are disclosed. These biosensors, which are dependent on the concentration/density of the BRET donor and acceptor in cellular compartments rather that specific protein-protein interactions, use a Renilla GFP/Luc BRET pair, which allows the robust and reproducible monitoring of protein trafficking/localization, with a sensitivity compatible with high-throughput screening (HTS). The use of these biosensors for various applications, including assessing/monitoring protein endocytosis, recycling and intracellular trafficking, receptor maturation/rescue by pharmacological chaperones, various endocytosis/exocytosis processes, activation/inhibition, as well as biomolecule concentration/density in different cellular compartments, is also disclosed.
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