Protein-protein interaction analysis using an affinity peptide tag and hydrophilic polystyrene plate.
A sandwich ELISA method using peptide tags showing a specific affinity to a hydrophilic polystyrene surface (PS-tags), PS 19 composed of RAFIASRRIKRP and KPS19R10 of KRAFIASRRIRRP and a hydrophilic polystyrene (phi-PS) plate was used to analyze protein-protein interactions. An Escherichia coli cysteine synthase complex, in which serine acetyltransferase (SAT) interacts with O-acetylserine sulfhydrylase-A (OASS) was used as a model system. When the interaction was detected by the conventional sandwich ELISA method using a hydrophobic polystyrene (pho-PS) plate, for the exclusive use of ELISA, the signal intensity was barely detectable due to conformational change of the ligand protein, OASS in the adsorbed state. On the contrary, when OASS, genetically fused with PS19 (OASS-PS19) or chemically conjugated with KPS19R10 (OASS-KPS19R10), was immobilized on the phi-PS plate, a high signal intensity was detected. Furthermore, by applying the two-step sandwich ELISA, in which OASS-PS19 or OASS-KPS19R10 formed a complex with SAT in the blocking solution before immobilization on the phi-PS plate, the signal intensity was further increased with a much shorter operational time, because SAT in the blocking solution formed a complex with OASS-PS19 or OASS-KPS19R10 without any steric hindrance.
Kumada
Yoichi
Y
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
Zhao
Chunhui
C
Ishimura
Ryota
R
Imanaka
Hiroyuki
H
Imamura
Koreyoshi
K
Nakanishi
Kazuhiro
K
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004800:Enzymes, Immobilized / Q000138:chemical synthesis*; D005982:Glutathione Transferase / Q000737:chemistry*; D007202:Indicators and Reagents; D010455:Peptides / Q000737:chemistry*; D011137:Polystyrenes; D011485:Protein Binding; D011487:Protein Conformation; D040901:Proteomics / Q000379:methods
D004800:Enzymes, Immobilized; D007202:Indicators and Reagents; D010455:Peptides; D011137:Polystyrenes; D005982:Glutathione Transferase