固定化金属亲和磁性纳米粒子的制备及其.doc

固定化金属亲和磁性纳米粒子的制备及其纯化组氨酸标签蛋白质的研究摘 要固定化金属亲和磁性纳米粒子（immobilized metal affinity magnetic nanoparticles, IMAN）纯化组氨酸标签蛋白技术近年来逐渐被关注。它结合了固定化金属亲和色谱（immobilized metal affinity chromatography, IMAC）和磁性纳米粒子（magnetic nanoparticles, MNPs）的优点，既能利用过渡金属离子Cu2+、Ni2+、Zn2+、Co2+等与氨基酸残基如组氨酸、半胱氨酸和色氨酸等的咪唑基、硫醇基和吲哚基的配位作用，选择性地纯化对金属离子有亲和力的蛋白质，又能充分发挥MNPs的磁性分离和高吸附容量优势，简单经济，快速高效。本论文研究了IMAN的关键技术配体的偶联和纯化条件，主要结果如下：1. IMAN的制备及表征采用碳二亚胺法将螯合配体IDA偶联到羧基化的磁性纳米粒子上，并进一步螯合过渡金属离子Cu2+、Ni2+、Zn2+或Co2+，制备出不同的IMAN。透射电镜显示偶联前后MNPs都是纳米尺寸，形貌无明显变化，偶联后MNPs仍保持了良好的分散性；红外光谱检测到偶联后MNPs的羰基峰比偶联前的明显增强，加入金属离子后，羰基峰向高频发生位移，表明有IDA偶联到羧基化MNPs上并成功螯合过渡金属离子；火焰原子吸收光谱法检测偶联IDA前后MNPs都能结合金属离子，但结合机理和稳定性还有待进一步研究。2. IMAN纯化组氨酸标签的蛋白质用所制备的IMAN纯化空载体pET-32c诱导表达的组氨酸标签蛋白质，SDS-聚丙烯酰胺凝胶电泳检测蛋白质纯度，UV法检测蛋白质浓度，主要研究结果如下： 研究组氨酸标签蛋白在IMAN上吸附量和洗脱量的影响因素，结论如下：吸附时间对吸附量有一定影响，45min时基本达到饱和吸附量；吸附时Tris缓冲液的pH值（7.1-8.9）对吸附量影响较大，pH较低时（7.1-7.4）吸附量大，当pH高于8.6时不利于吸附；咪唑洗脱液的浓度（0.2-1.0mol/L）对洗脱量有较大影响，0.8mol/L左右时，目标蛋白的洗脱量最大，过低和过高都不利于洗脱。 金属离子作为IMAN的螯合中心离子，其种类对组氨酸标签蛋白的吸附和洗脱影响较大。比较Cu2+、Ni2+、Zn2+和Co2+-IMAN对组氨酸标签蛋白的吸附量、洗脱量以及洗脱后IMAN上的剩留量，可得：吸附量大小依次为Cu2+Ni2+Zn2+Co2+；洗脱量依次为Zn2+Ni2+Cu2+Co2+；洗脱后IMAN上的剩留量为Cu2+Ni2+Zn2+Co2+。这与金属离子和蛋白质形成的配合物稳定性有关。对照组（无金属离子、无IDA和金属离子）也有少量目标蛋白被吸附和洗脱，甚至好于Co2+-IMAN。总之，Zn2+-IMAN更有利于纯化蛋白的回收。 通过L4(23)正交试验（金属离子：Cu2+、Zn2+；咪唑洗脱液浓度：0.5、1mol/L；重悬菌体Tris缓冲液pH值：7.1、8.0）得到的最佳纯化条件为：螯合中心离子为Zn2+、咪唑洗脱液浓度为1mol/L、重悬菌体Tris缓冲液pH值为7.1。关键词：金属螯合亲和；磁性纳米粒子；IMAN；制备；组氨酸标签；蛋白质纯化Preparation of immobilized metal affinity magnetic nanoparticles and its application to purification of His6-tagged proteinGuo Xiaoxiang (Biophysics)Directed by Prof. Yang WanshenAbstractImmobilized metal affinity magnetic nanoparticles (IMAN), a new technology for purification of His6-tagged protein, is being researched on the principle of immobilized metal affinity chromatography (IMAC). IMAN works fast and effectively not only because of the high affinity between transition metal ions Cu2+, Ni2+, Zn2+, Co2+ and the imidazolyl, thiol group, and indyl of histidine, cysteine, and tryptophane, but also because of its magnetic response and nano size which make purification process easier and adsorption capacity higher. There are two parts in this article:1. preparation and characterization of IMANThe IMAN is prepared by carboxyl-group functionalized magnetic nanoparticles as support, IDA as chelating legend and transition metal ions Cu2+, Ni2+, Zn2+ or Co2+ as chelating center ions. IDA is coupled to the carboxyl group by EDC and NHS.The TEM images show that theres no diffirences between nanoparticles with IDA and those without IDA. The FTIR spectra of nanoparticles indicates that IDA is coupled and transition metal ions Cu2+, Ni2+, Zn2+ and Co2+ are chelated on the IMAN respectively. The detection by FAAS also shows that metal ions are adsorbed by the nanoparticles with or without IDA but the reason is unknown.2. purification of His6-tagged proteinThe IMAN is used to purify His6-tagged protein. The protein purity and concentration are researched by SDS-PAGE and UV method. The results are as follows: There are several influencing factors to adsorption and elution. Binding time effects adsorption quantity and 45 min is the best one for adsorption quantity; the pH of binding Tris buffer (7.1-8.9) has a great effect on adsorption quantity: more His6-tagged protein is adsorbed when the pH is low (7.1-7.4) and when the pH is higher than 8.6, theres little adsorbed; the concentration of imidazole eluting buffer influences greatly on elution: the imidazole about 0.8 mol/L elutes more His6-tagged protein than those eluted by imidazole above or below 0.8 mol/L. Transition metal ions Cu2+, Ni2+, Zn2+ and Co2+ as chelating center ions have different abilities on binding and eluting. Compare the protein quantities and get the following results: for adsorption quantity of target protein, Cu2+Ni2+Zn2+Co2+; for elution, Zn2+Ni2+Cu2+Co2+; for remaining after eluting, Cu2+Ni2+Zn2+Co2+. It has a relation to the stability of the coordination compound. Some protein is adsorbed and eluted in the control groups (no metal ions and no IDA or metal ions), even more than the one using Co2+-IMAN. In a word, Zn2+-IMAN is better for the purification. L4 (23) orthogonal experiment is done (metal ions: Cu2+, Zn2+; the concentration of imidazole eluting buffer: 0.5, 1mol/L; the pH of binding Tris buffer: