Protein-protein interaction analysis using an affinity peptide tag and hydrophilic polystyrene plate.
Protein-protein interaction analysis using an affinity peptide tag and hydrophilic polystyrene plate.
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DOI：
10.1016/j.jbiotec.2006.09.018
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发表时间：
2007-02
影响因子：
4.1
通讯作者：
Y. Kumada;Chun-hui Zhao;Ryota Ishimura;H. Imanaka;K. Imamura;K. Nakanishi
中科院分区：
文献类型：
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作者：
Y. Kumada;Chun-hui Zhao;Ryota Ishimura;H. Imanaka;K. Imamura;K. Nakanishi
使用显示对亲水性聚苯乙烯表面（PS-标签）、由RAFIASRRIRRP和KRAFIASRRIRRP的KPS 19 R10组成的PS 19和亲水性聚苯乙烯（phi-PS）板的特异性亲和力的肽标签的夹心ELISA方法用于分析蛋白质-蛋白质相互作用。大肠杆菌半胱氨酸合酶复合物，其中丝氨酸乙酰转移酶（SAT）与O-乙酰丝氨酸巯基化酶-A（OASS）相互作用被用作模型系统。当通过使用疏水聚苯乙烯（pho-PS）板的常规夹心ELISA方法检测相互作用时，对于ELISA的专用，由于配体蛋白OASS在吸附状态下的构象变化，信号强度几乎检测不到。相反，当与PS19基因融合（OASS-PS19）或与KPS 19 R10化学缀合（OASS-KPS 19 R10）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-KPS 19 R10在固定在phi-PS板上之前与封闭溶液中的SAT形成复合物，信号强度在短得多的操作时间内进一步增加，因为封闭溶液中的SAT与OASS-PS19或OASS-KPS 19 R10形成复合物而没有任何空间位阻。
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.