【作者】 孙永亮；

【导师】 胡道道；

【作者基本信息】 陕西师范大学 ， 高分子化学与物理， 2007， 硕士

【摘要】 近年来，随着基因工程技术的发展，分离纯化基因重组蛋白的下游技术越来越显示出其重要性。从成分复杂的表达体系中分离纯化目的蛋白是一项艰巨而繁重的任务。目前基于分子的尺寸、形状、电荷、分子量、吸附等性质的差异进行蛋白质分离的方法也已在蛋白质纯化中得到不同程度的应用，而金属螯合亲和层析法（Metal Chelate Affinity Chromatographic，MCAC.又叫固定金属离子亲和层析法Immobilized Metal ion Affinity Chromatography，IMAC）以其分辨率高、选择性好、操作简单而成为一种重要的蛋白质分离方法。研究表明，影响金属螯合亲和色谱分离效果的因素很多，如溶液的pH值、盐浓度、洗脱方式等，但决定色谱填料分离效果的主要因素在于填料的基质、配基及螯合金属离子种类。琼脂糖（Agarose）具有良好的亲水性和稳定性，与可溶性蛋白的非特异性反应小。作为填料基质既可在非变性条件下使用，又可在变性条件（如6mol／L盐酸胍或8mol／L尿素）下使用。本实验用琼脂糖作为基质，选用四配位，与金属离子有较强作用力的羧甲基天冬氨酸为配基，以Co²⁺、Ni²⁺作为螯合金属离子，合成了一系列金属螯合亲和色谱填料，并对其蛋白质分离性能进行了研究。论文主要工作为以下几个方面：1．以琼脂糖（商品名SepharoseCl-6B）为基质，环氧氯丙烷为交连剂，以羧甲基天冬氨酸（CM-Asp）为配基制备了分别螯合Co²⁺和Ni²⁺的两种色谱介质：Agarose-CM-Asp-Co、Agarose-CM-Asp-Ni。对琼脂糖基质和Agarose-CM-Asp-Co、Agarose-CM-Asp-Ni的红外光谱表征结果表明：Co²⁺、Ni²⁺已经分别成功地螯合到被修饰的琼脂糖微球上。琼脂糖基质与Agarose-CM-Asp-Ni微球的电镜照片对比结果显示：介质表面光滑，说明在实验过程中没有引起微球形貌的变化，即在实验过程中没有产生能引起非特异性吸附的因素。用这两种介质对六聚组氨酸融合蛋白进行了分离，对目标蛋白的洗脱液做了SDS-PAGE分析，结果表明：两者都取得了良好的分离效果，并且Agarose-CM-Asp-Co分离效果最佳。2．以琼脂糖为基质，环氧氯丙烷为交联剂，制备了一系列不同羧甲基天冬氨酸担载量的介质微球：Agarose-0.5CM-Asp、Agarose-CM-Asp、Agarose-2CM-Asp。对其进行了滴定分析，测得各自的配基数目依次分别为：3.34×10^-5、3.83×10^-5、4.14×10^-5mol／L。结果表明：虽然配基CM-Asp的加入量成倍增加，但琼脂糖表面所能担载的配基CM-Asp的量增幅不大，说明在本实验条件下配基CM-Asp的螯合量已基本饱和。对上述三种不同CM-Asp担载量所制备螯合金属离子样品进行湿法硝解，通过原子吸收测定金属离子担载量，其结果也支持了上述观点。用上述三种不同CM-Asp担载量的微球所制备的金属离子Co²⁺、Ni²⁺螯合材料做六聚组氨酸融合蛋白分离，结果显示，金属离子螯合量的变化只影响到色谱介质的蛋白质吸附量，而对分离纯度没有明显影响。3．优化了对200μL细胞裂解液纯化所需的Agarose-CM-Asp-Co的介质用量、Agarose-CM-Asp-Co与细胞裂解液的孵育时间、介质的清洗条件、目标蛋白洗脱时所需咪唑浓度等条件。比较了Agarose-CM-Asp-Co与Ni-NTA-Agarose（Qiagen公司产品）两种螯合介质对融合蛋白的纯化效果。结果表明：针对200μL细胞裂解液的纯化体系，优选Co-CM-Asp-Sepharose（50％的悬浮液）的体积为60μL，最佳孵育时间为30min，洗脱时最佳咪唑浓度为200mM。与商品化的Ni-NTA-Agarose介质相比，清洗条件简单，介质选择性好，洗脱得到的靶蛋白纯度高。且本研究所得到的分离介质Agarose-CM-Asp-Co的分离性能优于与Qiagen公司产品Ni-NTA-Agarose。更多还原

【Abstract】 In recent years, with the development of techniques for geneticengineering, the techniques in separation recombination proteins become more andmore important. It is a hard and heavy task to isolate and purify proteins from thecomplicated expressed system. Based upon the difference in size, shape, charge,molecular weight and specific binding affinity of proteins, the methods for proteinseparation have been set up. Immobilized metal ion affinity chromatography （IMAC,also named metal chelate affinity chromatography） is one of the important methodsextensively used in separation of protein due to its high selectivity and simplicity.Researches indicate that there are many influence factors in the protein purificationprocess using IMAC, such as pH, the concentration of the salt, the way of elution, etc.However, the component of matrix, the ligand and the kinds of metal ions attached tothe matrix are the key factors. Agarose has some superior performance in hydrophilicity,stability, and nonspecific adsorption with soluble proteins. As support, Agarose can benot only used in normal condition, but also in denaturalization （for example, 6 mol/Lhydrochloric acid carbamidine or 8 mol/L urea）. Herein, the preparation of IMACmatrixes were conducted using agarose as support, carboxymethylated aspartate（CM-Asp） as chelating ligand and Co²⁺ and Ni²⁺ as chelating center ions. The mainworks include the following parts:1. IMAC matrixes named Agarose-CM-Asp-Co and Agarose-CM-Asp-Ni wereprepared using agarose as support, 3-Chloro-1,2-epoxy -propane as activated agent,carboxymethylated aspartate （CM-Asp） as chelating ligand, the Co²⁺ and Ni²⁺ as centerions. IR spectra of agarose, Agarose-CM-Asp-Co and Agarose-CM-Asp-Ni indicate thatCo²⁺ and Ni²⁺ were successfully chelated on the support respectively. SEM images ofagarose and Agarose-CM-Asp-Ni show that both kinds of microspheres are intact insurface. This feature of morphology is useful to reduce the nonspecific adsorption.Based on this IMAC matrixes, the purification of 6×His-tagged protein were conductedand the SDS-PAGE analysis to elution of target protein were also investigated. The results show that the matrix bonded with Co²⁺ is better than with Ni²⁺ in purification ofthe protein. Additionally, Agarose-CM-Asp-Co has high selectivity and low nonspecificabsoption.2. A series of mediums including Agarose-0.5CM-Asp、Agarose-CM-Asp andAgarose-2CM-Asp were prepared employing SepharoseC1-6B as support,3-Chloro-1,2-epoxypropane as activated agent, different amount of carboxymethylatedaspartate （CM-Asp） as chelating ligands. The amount of carboxyl goups activated in theprepared mediums is 3.34×10^-5, 3.83×10^-5 and 4.14×10^-5 mol/L, respectively. The resultindicates that the Agarose are saturated combined with CM-Asp in the given conditions.The amount of metal ion in Agarose-CM-Asp-M was determined by atoms absorptionspectra. The results testify the conclusion mentioned above. Agarose-CM-Asp-Mmediums with different amount of metal ions were used to purify 6×His-tagged proteinsand the results illustrate that the amount of metal ions in the mediums only influencesthe amount of chelated protein, but not the purity.3. The conditions for purification of 6×His-tagged protein including amount ofAgarose-CM-Asp-Co, the binding time between Agarose-CM-Asp-Co and lysate, thewashing condition and the imidazole concentration in the eluting buffer were optimized.The results show that 60μL （v/v 50% suspension） of Agarose-CM-Asp-Co solution issuitable for purification of the dissolved protein from 200μL of lysate, the optimalbinding time between Agarose-CM-Asp-Co and lysate is 30 min, and the optimalimidazole concentration in the eluting buffer is 200 mM. The efficiencies forpurification of 6×His-tagged proteins by Agarose-CM-Asp-Co and Ni-NTA-Agarosemade by Qiagen were compared. Compared with commercialized mediumNi-NTA-Agarose, the Agarose-CM-Asp-Co has prior selectivity and quality inpurification of 6×His-tagged proteins.更多还原