Coupling Recombinant PhyB to Cell Culture Plates and Beads

Area of Science:

• Biochemistry
• Molecular Biology
• Optogenetics

Background:

• Optogenetics offers precise control over biological processes using light.
• Phytochrome B (PhyB) is a plant photoreceptor that can be engineered for light-dependent interactions.

Purpose of the Study:

• To develop and validate a novel optogenetic system for controlling engineered receptors using PhyB.
• To enable high-spatiotemporal precision in studying receptor signaling pathways.

Main Methods:

• Utilized the N-terminal 651 amino acids of Arabidopsis thaliana PhyB as a light-sensitive ligand.
• Fused engineered receptors to Phytochrome-Interacting Factor 6 (PIF) for PhyB binding.
• Conjugated recombinant biotinylated PhyB to streptavidin-coated plates or beads.
• Validated the system using green fluorescent protein (GFP)-PIF-fused T-cell receptors (TCRs) in primary human T cells.

Main Results:

• Successfully coupled recombinant PhyB to plates and beads.
• Demonstrated light-dependent activation of PIF-fused receptors.
• Validated the optogenetic system's efficacy in primary human T cells.

Conclusions:

• The developed PhyB-based optogenetic system provides precise and reversible control over receptor activation.
• This method is suitable for studying cell-free interactions and receptor signaling with high spatiotemporal resolution.
• Offers a versatile tool for biological research and potential therapeutic applications.