Screening and characterization of affinity peptide tags specific to polystyrene supports for the orientated immobilization of proteins
Screening and characterization of affinity peptide tags specific to polystyrene supports for the orientated immobilization of proteins
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DOI：
10.1021/bp050331l
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发表时间：
2006-03-01
影响因子：
2.9
通讯作者：
Nakanishi, K
中科院分区：
文献类型：
--
作者：
Kumada, Y;Tokunaga, Y;Nakanishi, K
使用大肠杆菌随机肽展示文库系统筛选对聚苯乙烯表面表现出高亲和力特异性的十二肽（PS-标签），并将所述化合物用作肽标签用于蛋白质的位点特异性固定。10轮生物淘选后获得的各种PS-标签主要由碱性和脂肪族氨基酸残基组成，其中大部分排列在彼此接近。与所选PS标签融合的Muxylase型谷胱甘肽S-转移酶（GST）。当吸附在PS胶乳珠上时，在其C-末端的PS19（RAFIASRRIKRP）和PS23（AGLRLKKAAIHR），GST-PS19和GST-PS23具有比野生型GST更高的亲和力，并且固定化的突变型GST的比剩余活性比野生型GST高约10倍。使用对GST的N-末端肽特异性的抗肽抗体检测到的吸附在亲水和疏水PS表面上的GST-PS19和GST-PS23的信号强度远高于野生型GST的信号强度。这些发现表明，与选定的肽标签，PS19和PS23，融合的muplant型GST，可以位点特异性地固定在聚苯乙烯的表面上，其N-末端区域指向的解决方案。因此，所选择的肽标签将是有用的蛋白质固定在酶联免疫吸附测定（ELISA）系统和蛋白质为基础的生物芯片的建设。
Dodecapeptides that exhibit a high affinity specific to a polystyrene surface (PS-tags) were screened using an Escherichia coli random peptide display library system, and the compounds were used as a peptide tag for the site-specific immobilization of proteins. The various PS-tags obtained after 10 rounds of biopanning selection were mainly composed of basic and aliphatic amino acid residues, most of which were arranged in close proximity to one another. Mutant-type glutathione S-transferases (GSTs) fused with the selected PS-tags. PS19 (RAFIASRRIKRP) and PS23 (AGLRLKKAAIHR) at their C-terminus, GST-PS19 and GST-PS23, when adsorbed on the PS latex beads had a higher affinity than the wild-type GST, and the specific remaining activity of the immobilized mutant-type GSTs was approximately 10 times higher than that of the wild-type GST. The signal intensity detected for GST-PS19 and GST-PS23 adsorbed on hydrophilic and hydrophobic PS surfaces using an anti-peptide antibody specific for the N-terminus peptide of GST was much higher than that for the wild-type GST. These findings indicate that the mutant-type GSTs fused with the selected peptide tags, PS19 and PS23, could be site-specifically immobilized on the surface of polystyrene with their N-terminal regions directed toward the solution. Thus, the selected peptide tags would be useful for protein immobilization in the construction of enzyme-linked immunosorbent assay (ELISA) systems and protein-based biochips.