Highly-efficient purification of native polyhistidine-tagged proteins by multivalent NTA-modified magnetic nanoparticles
Highly-efficient purification of native polyhistidine-tagged proteins by multivalent NTA-modified magnetic nanoparticles
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DOI：
10.1021/bc060195l
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发表时间：
2007-03-01
影响因子：
4.7
通讯作者：
Lin, Wenbin
中科院分区：
文献类型：
--
作者：
Kim, Jason S.;Valencia, C. Alexander;Lin, Wenbin
以邻苯二酚为锚，合成了一种新的双氨三乙酸（NTA）螯合物，并将其固定在超顺磁性氧化铁纳米粒子上。当装载有Ni（II）时，这些双NTA固定的纳米颗粒显示出以其天然折叠构象结合多组氨酸（His x 6-标记的）融合蛋白，而商业微珠在相同条件下未能结合。用固定在氧化铁纳米颗粒上的单-NTA螯合物的对照实验表明对Hisx 6-标记的天然蛋白质具有类似的高亲和力，表明纳米颗粒呈现的高密度的单-NTA螯合物允许Hisx 6-标记与表面上的多于一个Ni-NTA部分结合。这项研究表明，多价策略可用于增强His x 6标记的蛋白质在其天然折叠构象中的结合。我们进一步证明了通过使用Ni（II）负载的氧化铁纳米颗粒从粗细胞裂解物中选择性纯化His x 6标记的蛋白质。本平台能够有效纯化在哺乳动物细胞中以低水平表达的Hisx 6标记的蛋白。因此，这项工作提出了一种新的基于纳米颗粒的高容量蛋白质纯化系统，与市售微珠相比，该系统具有更短的孵育时间，成比例的大洗涤量和显着更小的洗脱体积。
A new bis-nitrilotriacetic acid (NTA) chelate with catechol anchor was synthesized and immobilized on superparamagnetic iron oxide nanoparticles. When loaded with Ni(II), these bis-NTA-immobilized nanoparticles were shown to bind polyhistidine (His x 6-tagged) fusion proteins in their native, folded conformations that commercial microbeads failed to bind under identical conditions. Control experiments with a mono-NTA chelate immobilized on iron oxide nanoparticles indicate a similarly high affinity for His x 6-tagged native proteins, suggesting that the high density of the mono-NTA chelate presented by the nanoparticles allows the binding of the His x 6-tag to more than one Ni-NTA moiety on the surface. This study shows that the multivalency strategy can be utilized to enhance the binding of His x 6-tagged proteins in their native, folded conformations. We further demonstrated the selective purification of His x 6-tagged proteins from crude cell lysates by using the Ni(II)-loaded iron oxide nanoparticles. The present platform is capable of efficient purification of His x 6-tagged proteins that are expressed at low levels in mammalian cells. This work thus presents a novel nanoparticle-based high-capacity protein purification system with shorter incubation times, proportionally large washes, and significantly smaller elution volumes compared to commercially available microbeads.