[User Manual] PathHunter® Pharmacotrafficking Assays

[User Manual] PathHunter® Pharmacotrafficking Assays
Rev 4

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PathHunter Pharmacotrafficking cell lines are based on the enzymatic fragment complementation technology. The cells are expressed as a ProLink™ (PK)-tagged mutant transmembrane protein that is retained in the endoplasmic reticulum (ER) due to misfolding and co-expresses an Enzyme Acceptor (EA) tag localized either to (see figure below) (1) the cell membrane (Membrane-EA format), or (2) the early endosomes (Endosome-EA format). In either system, binding of a small molecule pharmacochaperone to the misfolded, PK-tagged protein stabilizes the protein in a conformation that enables its trafficking through the Golgi, then onward to the cell membrane. In the Membrane-EA format, complementation of the two β-galactosidase enzyme fragments (EA and PK) occurs at the membrane; while in the Endosome-EA format, protein re-localized to the cell membrane subsequently internalizes (either passively or actively) into endosomes, forcing complementation of EA-PK. The resulting functional, complemented β-galactosidase enzyme hydrolyzes substrate to generate a chemiluminescent signal.