Kumada Yoichi, Zhao Chunhui, Ishimura Ryota, Imanaka Hiroyuki, Imamura Koreyoshi, Nakanishi Kazuhiro
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
J Biotechnol. 2007 Feb 1;128(2):354-61. doi: 10.1016/j.jbiotec.2006.09.018. Epub 2006 Oct 19.
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.
一种使用对亲水性聚苯乙烯表面具有特异性亲和力的肽标签（PS标签）的夹心酶联免疫吸附测定（ELISA）方法，由RAFIASRRIKRP组成的PS 19以及KRAFIASRRIRRP的KPS19R10和亲水性聚苯乙烯（phi-PS）板用于分析蛋白质-蛋白质相互作用。以大肠杆菌半胱氨酸合酶复合物为模型系统，其中丝氨酸乙酰转移酶（SAT）与O-乙酰丝氨酸巯基酶-A（OASS）相互作用。当使用疏水性聚苯乙烯（pho-PS）板通过传统的夹心ELISA方法检测相互作用时，仅用于ELISA，由于吸附状态下配体蛋白OASS的构象变化，信号强度几乎无法检测到。相反，当与PS19基因融合（OASS-PS19）或与KPS19R10化学偶联（OASS-KPS19R10）的OASS固定在phi-PS板上时，检测到高信号强度。此外，通过应用两步夹心ELISA，其中OASS-PS19或OASS-KPS19R10在固定到phi-PS板之前在封闭溶液中与SAT形成复合物，信号强度进一步增加，操作时间更短，因为封闭溶液中的SAT与OASS-PS19或OASS-KPS19R10形成复合物时没有任何空间位阻。
