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首页> 外文期刊>Materials science & engineering, C. Materials for Biogical applications >[PHEMA/PEI]-Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma
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[PHEMA/PEI]-Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma

机译:[PHEMA / PEI] -Cu(II)固定化金属亲和层析冰冻凝胶:在从人血浆中分离IgG的应用

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摘要

The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]-Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50,100 and 150 mg/L. The separation of human IgG was achieved in one purification step at pH 7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]-Cu(II) (10% PEI) with 72.28 mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (C) 2016 Elsevier B.V. All rights reserved.
机译:固定化金属亲和色谱法(IMAC)作为治疗性蛋白质,核酸和其他生物分子的广泛分离和纯化工具已引起广泛关注。 IMAC对于具有天然表面暴露的组氨酸残基的蛋白质和具有组氨酸簇的重组蛋白质的巨大潜力。作为整体材料的冰晶石最近被提出作为用于下游工艺中生物分子分离的有前途的色谱吸附剂。在本研究中,已经合成了IMAC冷冻凝胶,并将其用于从IgG溶液和整个人血浆中吸附和分离免疫球蛋白G(IgG)。为此,使用聚(亚乙基亚胺)(PEI)作为螯合配体将Cu(II)离子与聚(甲基丙烯酸羟乙酯)PHEMA偶联。在这项研究中,通过改变PEI的比例(从1%到15%)以及不同量的作为螯合金属的Cu(II),可以优化冰晶的形成。通过扫描电子显微镜,傅立叶变换红外光谱和热重分析对制备的冰凝胶进行表征。分析[PHEMA / PEI] -Cu(II)冷冻凝胶结合水溶液中人IgG的能力。发现IMAC冷冻凝胶对人IgG具有高亲和力。用(10%PEI)研究了人IgG在PHEMA / PEI冷冻凝胶上的吸附,Cu(II)的浓度变化为10、50,100和150 mg / L。在一个纯化步骤中,pH 7.4即可完成人IgG的分离。在[PHEMA / PEI] -Cu(II)(10%PEI)和72.28 mg / g的人IgG处观察到最大吸附容量。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)研究了纯化效率和人IgG纯度。 (C)2016 Elsevier B.V.保留所有权利。

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