High biological activity of a recombinant protein immobilized onto polystyrene
High biological activity of a recombinant protein immobilized onto polystyrene
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DOI：
10.1002/biot.200800192
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发表时间：
2009-08-01
影响因子：
4.7
通讯作者：
Kishimoto, Michimasa
中科院分区：
文献类型：
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作者：
Kumada, Yoichi;Shiritani, Yuki;Kishimoto, Michimasa
研究了与聚苯乙烯（PS）结合肽（PS-标签）基因融合的谷胱甘肽 S-转移酶（GST）在亲水性增加的 PS 板上的吸附特性，以阐明 PS 标签融合蛋白与 PS 板之间特异性相互作用的机制。与 PS 标签 PS19 (RAFIASRRIKRP) 融合的 GST 优先与亲水性 PS 板相互作用，即使存在高浓度的竞争剂（例如 Tween 20 和 BSA）也是如此。 PS 标签中的碱性氨基酸和脂肪族氨基酸均参与 PS 标签与亲水性 PS 板表面的特异性相互作用。 PS19 变体 PS19-4 (RAIARRIRR) 和 PS19-6 (RIIIRRIRR) 的基因融合进一步提高了在高浓度竞争 BSA (50 mg/mL) 存在下 GST 的固定化产量。 PS19-6肽与各种亲水性PS板的表面发生特异性相互作用，尤其是在存在Tween 20的情况下。与野生型GST和GST-PS19相比，在所有固定有GST-PS19-6的亲水性PS板上检测到更高的剩余活性，并且通过在吸附状态下添加Tween 20进一步增加剩余活性。因此，本研究中开发的 PS19-6 肽作为亲和标签非常有用，可以将目标蛋白直接固定到各种亲水性 PS 支持物上，并具有高剩余活性。
The adsorption characteristics of glutathione S-transferases (GST) genetically fused with polystyrene (PS)-binding peptides (PS-tags) on PS plates with increase in hydrophilicity were studied to clarify the mechanisms of the specific interaction between the PS-tag-fused protein and PS plates. GST fused with the PS-tag PS19 (RAFIASRRIKRP) preferentially interacted with hydrophilic PS plates, even in the presence of high concentrations of competitors such as Tween 20 and BSA. Both basic and aliphatic amino acids in the PS-tags were involved in the specific interaction of PS-tags with the surface of the hydrophilic PS plate. Genetic fusion of the PS19 variants, PS19-4 (RAIARRIRR) and PS19-6 (RIIIRRIRR), further improved the immobilization yield of GST in the presence of a high concentration of the competitor BSA (50 mg/mL). The PS19-6 peptide specifically interacted with the surfaces of various hydrophilic PS plates, especially in the presence of Tween 20. Higher remaining activity was detected on all of the hydrophilic PS plates immobilized with GST-PS19-6 in comparison with those with wild-type GST and GST-PS19, and the remaining activity was further increased by the addition of Tween 20 in the adsorption state. The PS19-6 peptide developed in this study is therefore very useful as an affinity tag that can immobilize a target protein directly onto various hydrophilic PS supports with high remaining activity.