Dual-mode fluorophore-doped nickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site-specific fluorophore labeling
Dual-mode fluorophore-doped nickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site-specific fluorophore labeling
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DOI：
10.1021/ja074443f
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发表时间：
2007-10-31
影响因子：
15
通讯作者：
Katzenellenbogen, John A.
中科院分区：
文献类型：
--
作者：
Kim, Sung Hoon;Jeyakumar, M.;Katzenellenbogen, John A.
我们提出了第一个荧光团掺杂镍螯合物表面修饰二氧化硅纳米颗粒的例子，该纳米颗粒以双重模式发挥作用，将组氨酸标记的蛋白质纯化与位点特异性荧光团标记相结合。四甲基罗丹明 (TMR) 掺杂的二氧化硅纳米颗粒，估计每大约含有 700900 个 TMR。 23 nm 颗粒，用次氮基三乙酸 (NTA) 进行表面修饰，生成 TMR-SiO2-NTA-Ni2+。二氧化硅嵌入的 TMR 保留了非常高的量子产率，能够抵抗缓冲成分的猝灭，并且可以通过表面附着的 Ni2+ 适度猝灭且仅达到一定深度（约 2 nm）。当暴露于含有雌激素受体a的细菌裂解物时。配体结合域 (ER α) 作为次要成分，这些珠子显示出非常高的特异性结合，从而能够一步纯化蛋白质。通过电泳、辐射计数和 MALDI-TOF MS 表征这些珠子结合组氨酸标签蛋白的能力和特异性。 ER α 以位点特异性方式与 TMR-SiO2-NTA-Ni++ 珠结合，在溶液 FRET 实验以及蛋白质微阵列荧光和 FRET 测定中表现出良好的配体结合活性以及配体诱导的与共激活剂的结合活性。这种双模式型 TMR-SiO2-NTA-Ni2+ 系统代表了一步组氨酸标记蛋白纯化和多种荧光团种类的位点特异性标记的强大组合。
We present the first example of a fluorophore-doped nickel chelate surface-modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethy1rhodamine (TMR)-doped silica nanoparticles, estimated to contain 700900 TMRs per ca. 23 nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni2+. Silica-embedded TMR retains very high quantum yield, is resistant to quenching by buffer components, and is modestly quenched and only to a certain depth (ca. 2 nm) by surface -attached Ni2+. When exposed to a bacterial lysate containing estrogen receptor a. ligand binding domain (ER alpha) as a minor component, these beads showed very high specificity binding, enabling protein purification in one step. The capacity and specificity of these beads for binding a his-tagged protein were characterized by electrophoresis, radiometric counting, and MALDI-TOF MS. ER alpha, bound to TMR-SiO2-NTA-Ni++ beads in a site-specific manner, exhibited good activity for ligand binding and for ligand-induced binding to coactivators in solution FRET experiments and protein microarray fluorometric and FRET assays. This dual-mode type TMR-SiO2-NTA-Ni2+ system represents a powerful combination of one-step histicline-tagged protein purification and site-specific labeling with multiple fluorophore species.