Development and characterization of Ni-NTA-bearing microspheres
Development and characterization of Ni-NTA-bearing microspheres
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DOI：
10.1002/cyto.10124
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发表时间：
2002-07-01
期刊：
影响因子：
--
通讯作者：
Nolan, JP
中科院分区：
文献类型：
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作者：
Lauer, SA;Nolan, JP
背景资料：为了便于纯化，蛋白质通常用五个或六个相邻组氨酸残基（His-标签）的短亲和序列表达。该His-标签结合金属螯合剂络合物的金属，例如Ni 2 +-次氮基三乙酸（Ni-NTA）或-亚氨基二乙酸（Ni-IDA）。带有共价连接的金属螯合剂络合物的色谱树脂广泛用于His标记的蛋白质或肽的容易的亲和纯化。由于Ni-NTA微球在我们的研究开始时还没有商业化，我们制备并表征了这种微球以抑制His标记的蛋白质并研究它们的相互作用。我们的微球有三种类型：（a）共价结合到聚苯乙烯微球的金属螯合剂络合物，（B）共价结合到二氧化硅微球的金属螯合剂络合物，和（c）吸附到二氧化硅微球上形成自组装双层膜的脂质连接的金属螯合剂络合物，其中金属螯合剂具有横向移动性。通过将一种含伯胺的Ni-NTA配体与羧基化微球反应，然后负载Ni 2+，合成了一种负载Ni螯合剂的微球。通过将玻璃微球与含有磷脂酰胆碱（PC）和金属螯合脂质1，2-二油酰基甘油-3-[（N-（5-氨基-1-羧基戊基）亚氨基二乙酸）琥珀酰基]的脂质体孵育来制备具有横向移动的金属螯合剂的微球。His标记的增强型绿色荧光蛋白（EGFP）的结合被用来表征这些微球的特异性，灵敏度，容量和stability.Results流式细胞术：而所有micospheres特异性结合His标记的蛋白质，实现这一目标的条件是不同的聚苯乙烯和二氧化硅为基础的领域。所有三种类型的微球结合His-EGFP，在30-50 DM时发生饱和，在pH 7.4时表观亲合力（最大结合的一半浓度）约为1至2 X 10（-8）M。His-EGFP的结合被咪唑或乙二胺四乙酸（EDTA）抑制。聚苯乙烯镍-NTA微球表现出显着的非特异性结合，如通过在咪唑或EDTA的存在下结合或通过结合缺乏His-标签的荧光蛋白所测量的。这种蛋白质与聚苯乙烯球的非特异性结合和聚集只能通过包含低浓度的吐温20来防止，但不能通过包含牛血清白蛋白（BSA）、聚乙二醇或聚乙烯吡咯烷酮作为封闭剂来防止。相比之下，基于二氧化硅的微球与共价连接的Ni-NTA或二氧化硅微球承载吸附的双层，含有Ni-NTA-脂质在BSA的存在下表现出很少的非特异性结合。我们关于固定稳定性的结果表明，洗涤会使带有His标签的蛋白质与Ni-NTA微球的结合不稳定。这种结合由两种不同亲和力的相互作用组成。我们还表明，有限的多重分析与不同大小的二氧化硅微球轴承Ni-NTA-lipid是可行的。结论：所描述的微球是非常适合选择性地将His标记的蛋白质，通过流式细胞术分析它们的相互作用。His标记的蛋白质与Ni-NTA相互作用的亲和力和动力学稳定性不足以在多重分析形式中使用Ni-NTA微球，其中不同的His标记的蛋白质与不同的微球结合。为此目的的改进（改进的螯合剂和/或改进的亲和标签）对于扩展该方法的使用是关键的。我们目前正在研究新的螯合剂，以加强His标记的蛋白质固定到表面的稳定性。这种改进将大大增强固定化His标记蛋白质的相互作用的分析，并可能使基于微球的阵列与Histagged蛋白/抗体的发展成为可能。Cytometry 48：136-145，2002. (C)2002 Wiley-Liss，Inc.
Background: For ease of purification, proteins are often expressed with a short affinity sequence of five or six adjacent histidine residues (His-tag). This His-tag binds to the metal of metal chelator complexes such as Ni2+-nitrilotriacetic acid (Ni-NTA) or -iminodiacetic acid (Ni-IDA). Chromatography resins bearing covalently attached metal chelator complexes are used widely for the easy affinity purification of His-tagged proteins or peptides. Because Ni-NTA microspheres were not commercially available at the beginning of our studies, we prepared and characterized such microspheres to immobilize His-tagged proteins and study their interactions. Our microspheres are of three types: (a) metal chelator complexes bound covalently to polystyrene microspheres, (b) metal chelator complexes bound covalently to silica microspheres, and (c) lipid-linked metal chelator complexes adsorbed to silica microspheres forming self-assembled bilayer membranes where the metal chelators have lateral mobility.Methods: The microspheres bearing covalently attached Ni-chelator were synthesized by reacting a primary aminebearing Ni-NTA ligand with carboxy-functionalized microspheres and then loading with Ni2+. Microspheres with laterally mobile metal chelator were made by incubating glass microspheres with liposomes containing phosphatidylcholine (PC) and the metal chelating lipid 1,2-dioleoylsn-glvcero-3-[(N(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]. Binding of a His-tagged enhanced green fluorescent protein (EGFP) was used to characterize these microspheres by flow cytometry for their specificity, sensitivity, capacity and stability.Results: While all micospheres specifically bind His-tagged proteins, the conditions to achieve this are different for the polystyrene- and silica-based spheres. All three types of microspheres bind His-EGFP with saturation occurring at 30-50 DM and an apparent avidity (concentration of half maximal binding) of approximately 1 to 2 X 10(-8) M at pH 7.4. Binding of His-EGFP is inhibited by, imidazole or ethylene-diaminetetraacetic acid (EDTA). Polystyrene Ni-NTA microspheres showed significant nonspecific binding as measured by binding in the presence of imidazole or EDTA or by binding of fluorescent proteins lacking a His-tag. This nonspecific binding of proteins to and aggregation of polystyrene spheres could only be prevented by the inclusion of low concentrations of Tween 20, but not by including bovine serum albumin (BSA), polyethylene glycols, or polyvinylpyrrolidones as blocking agents. In contrast, silica-based microspheres with covalently attached Ni-NTA or silica microspheres bearing adsorbed bilayers that contain Ni-NTA-lipid showed little nonspecific binding in the presence of BSA. Our results on the stability of immobilization indicate that washing destabilizes the binding of His-tagged proteins to Ni-NTA microspheres. This binding consists of two interactions of different affinities. We also demonstrate that limited multiplexed analysis with differently sized silica microspheres bearing the Ni-NTA-lipid is feasible.Conclusions: The microspheres described are well suited to selectively immobilize His-tagged proteins to analyze their interactions by flow cytometry. The affinity and kinetic stability of the interaction of His-tagged proteins with Ni-NTA are insufficient to use Ni-NTA microspheres in multiplexed analysis formats where different His tagged proteins are bound to distinct microspheres. Improvements towards this end (improved chelators and/or improved affinity tags) are critical for extending the use of this method. We are currently working on novel chelators to strengthen the stability of immobilization of His-tagged proteins to surfaces. Such improvements would greatly enhance the analysis of interactions of immobilized His-tagged proteins and could make the development of microsphere-based arrays with Histagged protein/antibody possible. Cytometry 48:136-145, 2002. (C) 2002 Wiley-Liss, Inc.