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 receptor-ligand 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.
Shinya
Tomonori
T
Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan.
Osada
Tomohiko
T
Desaki
Yoshitake
Y
Hatamoto
Masahiro
M
Yamanaka
Yuko
Y
Hirano
Hisashi
H
Takai
Ryota
R
Che
Fang-Sik
FS
Kaku
Hanae
H
Shibuya
Naoto
N
D000345:Affinity Labels; D019921:Biotinylation; D002352:Carrier Proteins / Q000302:isolation & purification*; D003432:Cross-Linking Reagents; D018552:Daucus carota / Q000737:chemistry; D012275:Oryza / Q000737:chemistry; D010940:Plant Proteins / Q000302:isolation & purification*; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D000345:Affinity Labels; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D010940:Plant Proteins; D011956:Receptors, Cell Surface