Enhanced overexpression, purification of a channelrhodopsin and a fluorescent flux assay for its functional characterization

•A Pichia pastoris strain with enhanced overexpression of ChIEF-mCitrine is developed.•ATR is not required for proper trafficking of ChIEF-mCitrine and CaChR2 in yeast.•Light-responsive lipid nano-compartment is prepared using purified ChIEF-mCitrine.•A fluorescent flux assay for the protein’s light-sensory function is developed.•Adding ATR to purified ATR-free protein restores the protein function. Channelrhodopsins (ChRs) are a group of membrane proteins that allow cation flux across the cellular membrane when stimulated by light. They have been emerged as important tools in optogenetics where light is used to trigger a change in the membrane potential of live cells which induces downstream physiological cascades. There is also increased interest in their applications for generating light-responsive biomaterials. Here we have used a two-step screening protocol to develop a Pichia pastoris strain that produces superior yields of an enhance variant of CaChR2 (from Chlamydomonas reinhardtii), called ChIEF. We have also studied the effect of the co-factor, namely all-trans retinal (ATR), on the recombinant overexpression, folding, and function of the protein. We found that both ChIEF-mCitrine and CaChR2 can be overexpressed and properly trafficked to the plasma membrane in yeast regardless of the presence of the ATR. The purified protein was reconstituted into large unilamellar lipid vesicle using the detergent-assisted method. Using 9-amino-6-chloro-2-methoxyacridine (ACMA) as the fluorescent proton indicator, we have developed a flux assay to verify the light-activated proton flux in the ChIEF-mCitrine vesicles. Hence such vesicles are effectively light-responsive nano-compartments. The results presented in this work lays foundations for creating bio-mimetic materials with a light-responsive function using channelrhodopsins.