Coupling Recombinant PhyB to Cell Culture Plates and Beads

Optogenetics enables precise and reversible control of receptor activation using light-responsive proteins. In one approach, the first 651 amino acids of the photoreceptor phytochrome B (PhyB) of Arabidopsis thaliana are used as a light-responsive ligand for engineered receptors. These receptors are fused to the PhyB-interacting factor 6 (PIF) and bind to PhyB under red, but not under far-red light. The recombinant biotinylated PhyB ligand can be conjugated either to streptavidin-coated plates or to beads, enabling PIF-fused receptor activation with high valency. This method provides high spatiotemporal precision, offering potential for studying receptor signaling in two-dimensional cell-free interactions. Here, we provide protocols to couple recombinant PhyB to plastic plates or polystyrene beads and validate the coupling using light-dependent stimulation of green fluorescent protein (GFP)-PIF-fused T-cell receptors (TCRs) in primary human T cells.