Monitoring protein trafficking using beta-galactosidase reporter fragment complementation
View Patent ↗Methods and materials are disclosed for use in an enzyme fragment complementation assay using complementary fragments of β-galactosidase to study the trafficking of proteins in a cell. Compounds that bind to a target peptide have been found to affect protein folding and therefore trafficking. β-Galactosidase fragments, an enzyme donor (ED) and an enzyme acceptor (EA), are fused to a target peptide and to an intracellular compartment protein, wherein the compartment is involved in intracellular trafficking. Contacting the cell with a compound that binds to the target peptide results in enhanced movement of the protein through the cellular trafficking pathway comprised of the endoplasmic reticulum, Golgi apparatus, the plasma membrane, endosomes, etc. Using this approach, compounds that bind to a target peptide and alter its ability to traffic through the normal cellular pathway can be readily detected.
1. A method of determining an effect of a compound on trafficking of a target peptide, comprising:
a. providing a cell having an endoplasmic reticulum (“ER”), said cell comprising therein
i. a first fusion protein comprising the target peptide fused to a first β-galactosidase fragment wherein the target peptide has a mutation causing retention of the first fusion protein in the ER; and
ii. a second fusion protein comprising a protein localized to an endosome compartment fused to a second β-galactosidase fragment;
b. wherein said first and second β-galactosidase fragments have an affinity for each other to complement and produce an active β-galactosidase enzyme only when the first and second β-galactosidase units are in close proximity;
c. adding to said cell a compound that affects release of the target peptide from the ER; and
d. evaluating said cell for active β-galactosidase activity to determine whether the first and second fusion protein interact in said sub-cellular compartment as a result of release of the target peptide from the ER being affected by the compound.
2. The method of claim 1 wherein said cell is a mammalian cell.
3. The method of claims 1 wherein said target peptide is a cell membrane protein.
4. The method of claim 3 wherein said cell membrane protein is a G-protein coupled receptor (“GPCR”).
5. The method of claim 4 wherein the GPCR is one of a beta adrenergic receptor, histamine receptor, serotonin receptor, dopamine receptor, muscarinic receptor and angiotensin receptor.
6. The method of claim 1 wherein said first β-galactosidase fragment is an enzyme donor fragment.
7. The method of claim 1 wherein said second β-galactosidase fragment is localized to an endosome by a FYVE domain in said second fusion protein.
8. The method of claim 1 wherein the mutation is a single amino acid mutation.