混合模式高效液相色谱分离蛋白的基础研究及应用

发布时间：2017-12-27 09:04

  本文关键词：混合模式高效液相色谱分离蛋白的基础研究及应用　出处：《江南大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 混合模式 高效液相色谱 疏水电荷诱导色谱 保留因子 分离度

【摘要】：高效液相色谱(High performance liquid chromatography,HPLC)以分辨率高、分析速度快、重复性好的特点,而广泛运用于各个领域,成为色谱世界中的一个重要的组成部分。但是由于蛋白各个组份的性质相似,单一作用方式的技术还达不到分离纯度非常高的单晶蛋白质,限制了高效液相色谱在大分子领域的发展。因此,近些年来,多种不同的混合模式色谱(Mixed-mode chromatography,MMC)获得了大力的发展,疏水电荷诱导色谱(Hydrophobic charge induction chromatography,HCIC)就是其中之一。HCIC结合了疏水作用和静电相互作用,利用疏水作用进行吸附,电荷排斥作用进行解析,是一种混合型的双模式色谱技术。本文将常压下有着优异表现的HCIC引入到高效液相色谱中,形成一种新型的混合模式色谱技术即高效疏水电荷诱导色谱(High performance hydrophobic charge induction chromatography,HPHCIC),探索其用于蛋白的分离和分析。本文主要工作如下:(1)HPHCIC介质的制备。以硅烷偶联剂(KH560)为环氧活化剂,分别将2-巯基1-甲基咪唑、3-氨基吡啶、2-巯基苯并咪唑三种配基偶联到硅胶微球上(5μm,300?),制备了具有电荷作用力和疏水作用力的混合模式高效疏水电荷诱导色谱填料。环氧活化条件和配基偶联条件优化结果表明,在温度90℃、时间6 h条件下,KH560的活化密度最大,最大为165μmol?g-1;2-巯基1-甲基咪唑、2-巯基苯并咪唑、3-氨基吡啶三种配基最佳偶联密度分别为105μmol?g-1、45μmol?g-1、85μmol?g-1。(2)模型蛋白质在HPHCIC下的保留行为。以三甲基氯硅烷封尾的色谱柱为空白对照,考察三种模型蛋白(BSA,IgG,Lys)对三种HPHCIC介质上的相互作用。研究了流动相pH、流动相的盐离子浓度、流动相中乙腈百分含量对BSA、Lys和IgG三种蛋白的保留因子的影响。结果表明三种蛋白的保留行为受到了蛋白质和配基的疏水性质和电荷性质,以及流动相条件的综合影响,呈现一定的规律。三种配基的排斥力大小分别为3-氨基吡啶2-巯基1-甲基咪唑2-巯基苯并咪唑,而疏水大小为2-巯基苯并咪唑=2-巯基1-甲基咪唑3-氨基吡啶。3-氨基吡啶因为电荷排斥力过大,疏水作用较小,三种蛋白分离度差异小,似乎失去了选择能力,而2-巯基苯并咪唑为配基的HPHCIC介质则因为疏水作用力过大,BSA和IgG大部分都保留在色谱柱中。2-巯基1-甲基咪唑有丰富的配基密度,能够提供足够大的电荷排斥力,而且还具有适度的疏水性质,似乎最具备分离蛋白的HPHCIC介质。(3)HPHCIC分离模型蛋白研究。分别考察了三种色谱柱在流动相pH变化条件下对三种混合蛋白质的分离情况影响。发现3-氨基吡啶配基的HPHCIC介质出峰的峰面积大,但是分离度较小,说明了3-氨基吡啶作为配基确实有失去蛋白选择性的可能;2-巯基苯并咪唑虽然能够将三者分开,则因疏水作用过于强烈,导致BSA和IgG出峰面积小;2-巯基1-甲基咪唑的峰型较好,分离度也较高。选用2-巯基1-甲基咪唑配基作为HPHCIC填料进一步研究各种流动相对三种蛋白的分离度影响,通过不同的洗脱策略,发现低pH下的乙腈梯度对分离度的影响最大,而盐和pH梯度的存在并不能有效改变三种蛋白的分离度。三种蛋白质的分离行为的研究为以2-巯基1-甲基咪唑配基为HPHCIC填料的色谱柱应用提供了指导。(4)HPHCIC的应用研究:1、利用基因工程大肠杆菌进行Protein A蛋白的表达,离心破碎后收集上清液,预处理后进行Protein A的HPHCIC分离研究,发现目标蛋白的分离度较好。用Agilent C18色谱柱作为对照组,验证了HPHCIC的分离性能。2、利用HPHCIC进行蛋清溶菌酶的分析,同样用Agilent C18色谱柱作为对照组,也验证了HPHCIC的良好分离效果。
[Abstract]:High performance liquid chromatography (HPLC) has been widely applied in various fields with the characteristics of high resolution, fast analysis and good repeatability, and has become an important part of the chromatographic world. However, because of the similar properties of each component, the single action technology still can not reach the single crystal protein with high purity, which limits the development of high performance liquid chromatography in macromolecule field. Therefore, in recent years, Mixed-mode chromatography (MMC) has been developing vigorously, and Hydrophobic charge induction chromatography (HCIC) is one of them. HCIC combines hydrophobic interaction and electrostatic interaction, and adopts hydrophobic interaction and charge exclusion to analyze. It is a mixed mode of double mode chromatography. This paper will have excellent performance under atmospheric pressure HCIC is introduced into the high performance liquid chromatography, a new mixed mode chromatography is high performance hydrophobic charge induction chromatography (High performance hydrophobic charge induction to form chromatography, HPHCIC), to investigate the separation and analysis of proteins. The main work of this paper is as follows: (1) preparation of HPHCIC medium. Using silane coupling agent (KH560) for epoxy activator, respectively 2- mercapto 1- methyl imidazole, pyridine, 3- amino 2- mercaptobenzimidazoles three ligand coupling to the silica microspheres (5 m, 300?), the preparation of mixed mode charge charge with high performance hydrophobic force and hydrophobic forces induced by chromatography. Epoxy activation conditions and ligand coupling optimization results show that the temperature of 90 DEG C, time 6 h, the maximum density of activated KH560, the maximum is 165 mol? G-1; 2- mercapto 1- methyl imidazole, 2- 2-mercaptobenzimidazole and 3- aminopyridine ligands three best coupling density were 105 mol? G-1, 45 mol, 85 mol? G-1? G-1. (2) the retention behavior of the model protein under HPHCIC. In column three of methylchlorosilane sealing as blank control, inspection of the three model proteins (BSA, IgG, Lys) of three HPHCIC on the media interaction. The effects of the concentration of salt ions and the content of acetonitrile in the mobile phase on the retention factors of three proteins, BSA, Lys and IgG, were studied. The results showed that the retention behavior of the three proteins was affected by the hydrophobic properties and charge properties of the protein and ligand, and the combined effects of the mobile phase conditions. Three kinds of ligand repulsion size were 3- amino pyridine 2- mercapto 1- methyl imidazole 2- MERCAPTOBENZIMIDAZOLE, while the hydrophobic size 2- mercaptobenzimidazoles =2- mercapto 1- methyl imidazole 3- amino pyridine. 3- aminopyridine because the charge repulsion force is too large, the hydrophobic effect is small, three kinds of protein separation degree difference is small, seems to have lost the ability to 2- and mercaptobenzimidazoles HPHCIC ligand is because the hydrophobic force is too large, most of the BSA and IgG are retained in the column. 2- mercapto 1- methyl imidazole has abundant ligand density, which can provide enough charge repulsion, and also has moderate hydrophobic property. It seems to have the HPHCIC medium with the most protein separation. (3) the study of HPHCIC separation model protein. The effects of three chromatographic columns on the separation of three kinds of mixed proteins under the changing conditions of mobile phase pH were investigated. 3- amino pyridine ligand HPHCIC medium peak area peak, but the degree of separation of small, shows 3- amino pyridine as ligand protein may indeed lose selectivity; 2- MERCAPTOBENZIMIDAZOLE is able to separate the three, due to the hydrophobic interaction is too strong, leading to BSA and IgG peak area is small; the peak type 2- mercapto 1- methyl imidazole is better, higher degree of separation. Use 2- mercapto 1- methyl imidazole ligand HPHCIC as filler for further studies of effect of relative flow separation of three proteins, the strategy of different elution gradient, was found at low pH have the greatest influence on the degree of separation, and the salt and pH gradient does not effectively change the degree of separation of three proteins. The study of the separation behavior of the three proteins provides guidance for the application of the 2- mercapto 1- methyl imidazole ligand as the HPHCIC filler. (4) the application of HPHCIC: 1. The expression of Protein A protein was detected by genetic engineering E.coli. After centrifugation, the supernatant was collected. After pretreatment, the HPHCIC separation of Protein A was carried out, and the target protein was well separated. The Agilent C18 chromatographic column was used as the control group to verify the separation performance of HPHCIC. 2. The analysis of egg white lysozyme was carried out by HPHCIC, and the Agilent C18 column was also used as the control group, and the good separation effect of HPHCIC was also verified.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O652.63;O629.73
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本文编号：1341056