GFP fusion proteins and their use
View Patent ↗The present invention provides fusion proteins including a green fluorescent protein inserted into the internal amino acid sequence of a Gαs protein and further provides method of using the fusion protein construct to follow activation of a G-protein receptor by a candidate drug.
1. A fusion protein comprising a green fluorescent protein inserted between the GTPase and the helical domain of Gα S , wherein the Gα S activates adenylyl cyclase and the fusion protein translocates from the plasma membrane into the cytoplasm upon activation.
2. The fusion protein of claim 1 , wherein the insertion is at regions that are free of interactions with receptors or effectors.
3. The fusion protein of claim 1 modified for specific receptors by replacing amino acid residues at the C terminal end of Gα s .
4. A method for making a fusion protein, said method comprising:
(a) obtaining a molecule having the amino acid sequence of a green fluorescent protein; and
(b) inserting the molecule between the GTPase and the helical domain of Gα S .
5. The method of claim 4 wherein the fusion protein has the amino acid sequence of SEQ ID NO: 2.
6. A method to follow an activation of a G-protein receptor by a candidate drug said method comprising:
(a) obtaining a Gα s green fluorescent fusion protein, wherein the green fluorescent protein is inserted between the GTPase and the helical domain of Gα S ;
(b) monitoring fluorescence of the fusion protein in response to the candidate drug;
(c) inferring from a change in membrane fluorescence whether the drug is an agonist or antagonist; and
(d) following the activation of the G-protein receptor by the candidate drug.
7. The fusion protein of claim 1 comprising the amino acid sequence of SEQ ID NO: 2.
8. The fusion protein of claim 1 , wherein the fusion protein internalizes.