Characterization of Receptor Proteins using Affinity Cross-linking with Biotinylated Ligands

The plant genome encodes a wide range of receptor-like proteins but the function of most of these proteins is unknown. We propose the use of affinity cross-linking of biotinylated ligands for a ligand-based survey of the corresponding receptor molecules. Biotinylated ligands not only enable the analysis of receptorligand interactions without the use of radioactive compounds but also the isolation and identification of receptor molecules by a simple affinity trapping method. We successfully applied this method for the characterization, isolation and identification of the chitin elicitor binding protein (CEBiP). A biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the detection of CEBiP in the plasma or microsomal membrane preparations from rice and carrot cells. Binding characteristics of CEBiP analyzed by inhibition studies were in good agreement with the previous results obtained with the use of a radiolabeled ligand. The biotin-tagged CEBiP could be purified by avidin affinity chromatography and identified by LC-MALDI-MS/MS after tryptic digestion. We also used this method to detect OsFLS2, a rice receptor-like kinase for the perception of the peptide elicitor flg22, in membrane preparations from rice cells overexpressing OsFLS2. This work demonstrates the applicability of this method to the purification and identification of plant receptor proteins.