本文關(guān)鍵詞：混合模式高效液相色譜分離蛋白的基礎(chǔ)研究及應(yīng)用　出處：《江南大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 混合模式 高效液相色譜 疏水電荷誘導(dǎo)色譜 保留因子 分離度

【摘要】：高效液相色譜(High performance liquid chromatography,HPLC)以分辨率高、分析速度快、重復(fù)性好的特點(diǎn),而廣泛運(yùn)用于各個(gè)領(lǐng)域,成為色譜世界中的一個(gè)重要的組成部分。但是由于蛋白各個(gè)組份的性質(zhì)相似,單一作用方式的技術(shù)還達(dá)不到分離純度非常高的單晶蛋白質(zhì),限制了高效液相色譜在大分子領(lǐng)域的發(fā)展。因此,近些年來,多種不同的混合模式色譜(Mixed-mode chromatography,MMC)獲得了大力的發(fā)展,疏水電荷誘導(dǎo)色譜(Hydrophobic charge induction chromatography,HCIC)就是其中之一。HCIC結(jié)合了疏水作用和靜電相互作用,利用疏水作用進(jìn)行吸附,電荷排斥作用進(jìn)行解析,是一種混合型的雙模式色譜技術(shù)。本文將常壓下有著優(yōu)異表現(xiàn)的HCIC引入到高效液相色譜中,形成一種新型的混合模式色譜技術(shù)即高效疏水電荷誘導(dǎo)色譜(High performance hydrophobic charge induction chromatography,HPHCIC),探索其用于蛋白的分離和分析。本文主要工作如下:(1)HPHCIC介質(zhì)的制備。以硅烷偶聯(lián)劑(KH560)為環(huán)氧活化劑,分別將2-巰基1-甲基咪唑、3-氨基吡啶、2-巰基苯并咪唑三種配基偶聯(lián)到硅膠微球上(5μm,300?),制備了具有電荷作用力和疏水作用力的混合模式高效疏水電荷誘導(dǎo)色譜填料。環(huán)氧活化條件和配基偶聯(lián)條件優(yōu)化結(jié)果表明,在溫度90℃、時(shí)間6 h條件下,KH560的活化密度最大,最大為165μmol?g-1;2-巰基1-甲基咪唑、2-巰基苯并咪唑、3-氨基吡啶三種配基最佳偶聯(lián)密度分別為105μmol?g-1、45μmol?g-1、85μmol?g-1。(2)模型蛋白質(zhì)在HPHCIC下的保留行為。以三甲基氯硅烷封尾的色譜柱為空白對照,考察三種模型蛋白(BSA,IgG,Lys)對三種HPHCIC介質(zhì)上的相互作用。研究了流動(dòng)相pH、流動(dòng)相的鹽離子濃度、流動(dòng)相中乙腈百分含量對BSA、Lys和IgG三種蛋白的保留因子的影響。結(jié)果表明三種蛋白的保留行為受到了蛋白質(zhì)和配基的疏水性質(zhì)和電荷性質(zhì),以及流動(dòng)相條件的綜合影響,呈現(xiàn)一定的規(guī)律。三種配基的排斥力大小分別為3-氨基吡啶2-巰基1-甲基咪唑2-巰基苯并咪唑,而疏水大小為2-巰基苯并咪唑=2-巰基1-甲基咪唑3-氨基吡啶。3-氨基吡啶因?yàn)殡姾膳懦饬^大,疏水作用較小,三種蛋白分離度差異小,似乎失去了選擇能力,而2-巰基苯并咪唑?yàn)榕浠腍PHCIC介質(zhì)則因?yàn)槭杷饔昧^大,BSA和IgG大部分都保留在色譜柱中。2-巰基1-甲基咪唑有豐富的配基密度,能夠提供足夠大的電荷排斥力,而且還具有適度的疏水性質(zhì),似乎最具備分離蛋白的HPHCIC介質(zhì)。(3)HPHCIC分離模型蛋白研究。分別考察了三種色譜柱在流動(dòng)相pH變化條件下對三種混合蛋白質(zhì)的分離情況影響。發(fā)現(xiàn)3-氨基吡啶配基的HPHCIC介質(zhì)出峰的峰面積大,但是分離度較小,說明了3-氨基吡啶作為配基確實(shí)有失去蛋白選擇性的可能;2-巰基苯并咪唑雖然能夠?qū)⑷叻珠_,則因疏水作用過于強(qiáng)烈,導(dǎo)致BSA和IgG出峰面積小;2-巰基1-甲基咪唑的峰型較好,分離度也較高。選用2-巰基1-甲基咪唑配基作為HPHCIC填料進(jìn)一步研究各種流動(dòng)相對三種蛋白的分離度影響,通過不同的洗脫策略,發(fā)現(xiàn)低pH下的乙腈梯度對分離度的影響最大,而鹽和pH梯度的存在并不能有效改變?nèi)N蛋白的分離度。三種蛋白質(zhì)的分離行為的研究為以2-巰基1-甲基咪唑配基為HPHCIC填料的色譜柱應(yīng)用提供了指導(dǎo)。(4)HPHCIC的應(yīng)用研究:1、利用基因工程大腸桿菌進(jìn)行Protein A蛋白的表達(dá),離心破碎后收集上清液,預(yù)處理后進(jìn)行Protein A的HPHCIC分離研究,發(fā)現(xiàn)目標(biāo)蛋白的分離度較好。用Agilent C18色譜柱作為對照組,驗(yàn)證了HPHCIC的分離性能。2、利用HPHCIC進(jìn)行蛋清溶菌酶的分析,同樣用Agilent C18色譜柱作為對照組,也驗(yàn)證了HPHCIC的良好分離效果。
[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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：O652.63;O629.73
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本文編號：1341056