铁镍磁性微球直接纯化组氨酸标签蛋白
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摘要
以氯化铁与硫酸镍为主要原料基于热分解法原理一锅法合成铁镍磁性微球。氯化铁、硫酸镍和乙酸钠在(80℃)乙二醇中搅拌30 min,在氮气保护下,加入乙醇胺后180℃搅拌6h,经过水清洗与乙醇清洗后获得有磁性的含镍微球,XRD检测结果证实晶型为Ni Fe2O4,晶体的平均粒径为6.75 nm。磁性微球不经任何修饰直接与组氨酸标签蛋白结合,完成带有组氨酸标签的绿色荧光蛋白纯化,SDS-PAGE检测结果表明:使用5倍于镍配位凝胶质量的铁镍磁性微球可达到与镍配位凝胶相近的蛋白质吸附量。低成本的铁镍磁性微球可以替代镍配位凝胶用于组氨酸标签蛋白的分离纯化。
The iron-nickel magnetic nanoparticle was synthesized with thermal decomposition method. Ferric chloride,nickel sulfate and sodium acetate were stirred in ethylene glycol at 80℃ for 30 minutes,and then stirred at 180℃ under the protection of nitrogen for 6 hours after the addition of ethanol amine,the iron-nickel magnetic nanoparticles were obtained with one pot method and were washed with water and ethanol. The nanoparticles could be dispersed in water and gathered in magnetic field. According to the XRD spectrum and the standard database,these results were obtained: the magnetic particle was cube,the crystal type was Ni Fe2O4,and the particle size was 6. 75 nm calculated with Scherrer formal. The His-tagged green fluorescent protein( GFP) was expressed in E-. coli under the induction of IPTG. The supernatant from the ultrasonic lysates was associated with the magnetic nanoparticles to purify the tagged GFP. The GFP was obtained with different elution buffers from the magnetic nanoparticles or Ni-NTA agarose and detected with SDS-PAGE. The results showed that the protein yield of Ni-NTA agarose was five times than the magnetic nanoparticles. The low-cost nickel-iron magnetic nanoparticles were obtained by the hydrothermal synthesis method,and could replace the NiNTA agarose to purify the tagged green fluorescent protein directly.
The iron-nickel magnetic nanoparticle was synthesized with thermal decomposition method. Ferric chloride,nickel sulfate and sodium acetate were stirred in ethylene glycol at 80℃ for 30 minutes,and then stirred at 180℃ under the protection of nitrogen for 6 hours after the addition of ethanol amine,the iron-nickel magnetic nanoparticles were obtained with one pot method and were washed with water and ethanol. The nanoparticles could be dispersed in water and gathered in magnetic field. According to the XRD spectrum and the standard database,these results were obtained: the magnetic particle was cube,the crystal type was Ni Fe2O4,and the particle size was 6. 75 nm calculated with Scherrer formal. The His-tagged green fluorescent protein( GFP) was expressed in E-. coli under the induction of IPTG. The supernatant from the ultrasonic lysates was associated with the magnetic nanoparticles to purify the tagged GFP. The GFP was obtained with different elution buffers from the magnetic nanoparticles or Ni-NTA agarose and detected with SDS-PAGE. The results showed that the protein yield of Ni-NTA agarose was five times than the magnetic nanoparticles. The low-cost nickel-iron magnetic nanoparticles were obtained by the hydrothermal synthesis method,and could replace the NiNTA agarose to purify the tagged green fluorescent protein directly.
引文
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[3]曹孟菁,朱维耀,蔡强,等.可控粒径PAA/Fe3O4磁性颗粒的制备[J].功能材料,2015,07:7103-7108.
[4]张恒頔,胡立立,钟毅,等.单分散性PLGA磁性微球的制备及其缓释性[J].精细化工,2015,03:267-271+276.
[5]王祝敏,张凤霞,马云龙.Fe3O4/聚苯乙烯-丙烯酸磁性微球的制备与表征[J].化工新型材料,2014,12:192-194.
[6]黄华斌,庄峙厦,杨朝勇,等.表面电性可控的磁性微球的制备、表征及其在基因组DNA萃取中的应用[J].分析化学,2014,11:1598-1603.
[7]王文加,郭晓林,韦安慧,等.基于Fe3O4@Si O2/Ni-NTA磁性微球的His-tag融合蛋白纯化体系的建立[J].高等学校化学学报,2012,02:303-307.
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