{"pk":4847,"title":"Determination of Immune Signal–Receptors, PD-1 and PD-L1, Interactions in Solution using qFRET Technology","subtitle":null,"abstract":"Human PD-L1 (programmed cell death 1 - ligand 1) is a transmembrane protein that is highlyexpressed on the membrane of cancer cells. It binds to inhibitory receptor PD-1 (programmed celldeath protein - 1), which is expressed on the surface of cytotoxic T cells. The interaction betweenPD-L1 and PD-1 reduces the effect of anti-tumor immune response and the strategy of blocking theirinteraction has been used for anti-cancer drug manufacture. Past studies isolated the extracellulardomain of PD-L1 for characterization of the structure. This study aims to recover, isolate, and purifythe insoluble PD-L1 protein (external domain), and study its binding interaction with PD-1 forthe development of an in vitro quantitative FRET (qFRET) assay. To report PD-L1/PD-1 bindinginteraction, fluorescent donor and acceptor pairs, CyPet and YPet were bound to PD-L1 and PD-1proteins respectively and qFRET was applied to assess the interaction of the two proteins basedentirely on fluorescence.The results provide evidence of recovery of the purified and refolded CyPet-Ext.PDL1 and YPet-Ext.PD1 proteins from cell pellets, shown in the coomassie stained SDS-PAGE gel. This study alsoshows that the proteins were able to be recovered through affinity chromatography under denaturingconditions. The qFRET technique showed that the acceptor, YPet-Ext.PD1, is interacting with thedonor, CyPet-Ext.PDL1. This study provides a novel method for better understanding the bindingmechanism of PD-L1/PD-1 that can be applied to other cell-surface protein interactions, as well as tostipulate a platform for small molecule inhibitor related drug screenings and production.","language":"en","license":null,"keywords":[{"word":"PD-1"},{"word":"PD-L1"},{"word":"Expression"},{"word":"Immunotherapy"},{"word":"Antibody"},{"word":"FRET"}],"section":"Articles","is_remote":true,"remote_url":"https://escholarship.org/uc/item/2d09435x","frozenauthors":[{"first_name":"Mary","middle_name":"","last_name":"Nwangwu","name_suffix":"","institution":"","department":"None"},{"first_name":"Jiayu","middle_name":"","last_name":"Liao","name_suffix":"","institution":"","department":"None"}],"date_submitted":"2018-05-24T22:43:34Z","date_accepted":"2018-05-24T22:43:34Z","date_published":"2018-01-01T00:00:00Z","render_galley":null,"galleys":[{"label":"","type":"","path":"https://journalpub.escholarship.org/ucr_undergrad_research_j/article/4847/galley/2742/download/"}]}