A nickel chelate microtiter plate assay for six histidine-containing proteins
A nickel chelate microtiter plate assay for six histidine-containing proteins
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DOI：
10.1006/abio.1996.0050
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发表时间：
1996-02-01
影响因子：
2.9
通讯作者：
Dougherty, JP
中科院分区：
文献类型：
--
作者：
Paborsky, LR;Dunn, KE;Dougherty, JP
通过使用亲和标记物，蛋白质纯化已经变得明显更容易，所述亲和标记物可以在重组蛋白的氨基末端或羧基末端进行遗传工程改造。最广泛使用的标签之一是六个连续的组氨酸残基或6 His标签。这些残留物即使在变性剂存在下也能以高亲和力与固定在螯合树脂上的金属离子结合，并且可以用咪唑温和洗脱。我们报告的方法固定化的六个含组氨酸的蛋白质到微量滴定板。合成了一种氨三乙酸衍生物。这种衍生物，N，N-双[羧甲基]赖氨酸（BCML），很容易耦合到马来酸酐去活化的聚苯乙烯微量滴定板。然后用Ni 2+填充板以捕获6 His标记的蛋白质。使用两种不同的重组蛋白与6 His标签在N-或C-末端，我们证明了结合到Ni 2 +-NTA板是特异性的六个含组氨酸的蛋白质。缺乏6 His标签的蛋白质不与板结合。该板用于改良的酶联免疫吸附测定形式，以定量蛋白质浓度并测定蛋白质-配体相互作用的亲和力。该技术还可以扩展到包括蛋白质-蛋白质相互作用的拮抗剂的高通量筛选测定。(C)出版社：Academic Press，Inc.
Protein purification has been made significantly easier by the use of affinity tags that can be genetically engineered at either the amino- or carboxyl-terminus of recombinant proteins. One of the most widely used tags is six consecutive histidine residues or 6His tag. These residues bind with high affinity to metal ions immobilized on chelating resins even in the presence of denaturing agents and can be mildly eluted with imidazole. We report the methodology for the immobilization of six histidine-containing proteins onto microtiter plates. A derivative of nitrilotriacetic acid (NTA) was prepared. This derivative, N,N-bis[carboxymethyl]lysine (BCML), was easily coupled to a maleic anhydride-activated polystyrene microtiter plate. The plate was then charged with Ni2+ for the capture of the 6His-tagged proteins. Using two different recombinant proteins with the 6His tag at either the N- or C-terminus, we demonstrated that the binding to the Ni2+-NTA plate was specific for six histidine-containing proteins. Proteins lacking the 6His tags did not bind to the plate. The plate was used in a modified enzyme-linked immunoabsorbent assay format to quantitate protein concentrations and determine the affinity of protein-ligand interactions. The technology can also be extended to include high-throughput screening assays for antagonists of protein-protein interactions. (C) 1996 Academic Press, Inc.