Sigal G B, Bamdad C, Barberis A, Strominger J, Whitesides G M
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138, USA.
Anal Chem. 1996 Feb 1;68(3):490-7. doi: 10.1021/ac9504023.
This paper reports the generation of a self-assembled monolayer (SAM) that selectively binds proteins whose primary sequence terminates with a His-tag: a stretch of six histidines commonly incorporated in recombinant proteins to simplify purification. The SAM was prepared by the adsorption onto a gold surface of a mixture of two alkanethiols: one thiol that terminated with a nitrilotriacetic acid (NTA) group, a group that forms a tetravalent chelate with Ni(II), and a second thiol that terminated with a tri(ethylene glycol) group, a group that resists protein adsorption. His-tagged proteins bound to the SAM by interaction of the histidines with the two vacant sites on Ni(II) ions chelated to the surface NTA groups. Studies with model proteins showed the binding was specific for His-tagged proteins and required the presence of Ni(II) on the surface. Immobilized His-tagged proteins were kinetically stable in buffered saline at pH 7.2 but could be desorbed by treatment with 200 mM imidazole. Surface plasmon resonance studies for two model systems showed that His-tagged proteins adsorbed on the NTA-SAM retained a greater ability to participate in binding interactions with proteins in solution than protein immobilized in a thin dextran gel layer by covalent coupling.
本文报道了一种自组装单分子层（SAM）的生成，该单分子层能选择性结合其一级序列以His标签结尾的蛋白质：一段通常包含在重组蛋白中以简化纯化过程的六个组氨酸序列。该SAM是通过将两种链烷硫醇的混合物吸附到金表面制备而成：一种硫醇以次氮基三乙酸（NTA）基团结尾，该基团能与Ni（II）形成四价螯合物；另一种硫醇以三（乙二醇）基团结尾，该基团能抵抗蛋白质吸附。带有His标签的蛋白质通过组氨酸与螯合到表面NTA基团上的Ni（II）离子的两个空位相互作用而与SAM结合。对模型蛋白的研究表明，这种结合对带有His标签的蛋白具有特异性，并且表面需要存在Ni（II）。固定化的带有His标签的蛋白在pH 7.2的缓冲盐溶液中动力学稳定，但可用200 mM咪唑处理使其解吸。对两个模型系统的表面等离子体共振研究表明，与通过共价偶联固定在薄葡聚糖凝胶层中的蛋白质相比，吸附在NTA-SAM上的带有His标签的蛋白质保留了更强的与溶液中蛋白质参与结合相互作用的能力。
