一种疏水色谱填料的特性及应用的研究

 以交联壳聚糖为基质 ,正戊醛为配基 ,利用改进的方法制备了疏水作用色谱 (HIC)填料 ,并对该色谱填料的吸附行为和应用作了研究。结果表明 ,此类填料对蛋白质的吸附行为符合疏水相互作用理论 ,对α 淀粉酶的纯化活性回收率大于 80 %。     

氨基己酸为配基的新型弱阳离子交换/疏水双功能色谱固定相对蛋白质的分离纯化

以硅胶为基质、氨基己酸为配基制备了一种新型弱阳离子交换/疏水(WCX/HIC)双功能混合模式色谱固定相。该固定相配基具有一定的疏水性且含有羧基,在高盐浓度下表现为HIC的性质,可作为HIC固定相使用;在低盐浓度条件下表现为离子交换的性质,可作为WCX固定相使用。分别考察了该介质在WCX和HIC两种模式下对标准蛋白质的分离性能,并与商品柱进行比较。结果表明,所合成的WCX/HIC双功能固定相在WCX和HIC两种模式下对蛋白质均有较高的分离度和选择性,且分离能力与商品柱相当,两种模式下标准蛋白质的质量和活性回收率均大于93%,表明该柱具有"一柱二用"的功能,适于生物大分子的分离纯化。基于此双功能色谱柱构建的在线单柱二维液相色谱(2DLC-1C)可在60 min内实现8种蛋白质的快速分离。在70 min内完成了对蛋清中溶菌酶的二维纯化,纯度可达到98.3%。该技术中一根色谱柱可当作两根色谱柱使用,对蛋白质组学研究和重组蛋白药物的生产具有重要的应用价值。     

高效疏水作用色谱填料的合成及其在重组人干扰素纯化中的应用

高效疏水作用色谱（HPHIC）是利用不同蛋白质表面疏水区域与填料之间具有不同疏水作用进行分离的．由于HPHIC采用盐水体系作为流动相，配体采用极性的有机基团，使蛋白质可以在十分温和的条件下进行分离，且保持其生物活性基本不变[1，2]．自80年代中期以来，HPHIC在蛋白质的分离纯化上得到了广泛的应用．在90年代初期，随着基因工程技术的发展，HPHIC同时也被应用到基因工程的下游纯化技术上[3，4]．本文中我们合成了一OCH2CH3为端基的填料，检验了该填料的分离效果，并利用该填料对酵母菌表达的人αA-干扰素、大肠杆菌（E．col…     

动力学因素对液相色谱分离整体蛋白的影响

依据液相色谱分离整体蛋白的效果与色谱柱柱长基本无关的事实,研究了动力学因素对疏水相互作用色谱(HIC)分离整体蛋白的影响。首次提出了用于线性梯度洗脱条件下蛋白分离的“条件板高”(H)概念,并将其用于动力学因素对分离整体蛋白的影响的表征。分别用常用的色谱柱和色谱饼对标准蛋白进行了分离,绘制了类似于van Deemter的“条件板高”对流动相线速(u)的曲线图。发现对应于色谱柱最低“条件板高”的适合线速约为色谱饼的1/5～1/15,且色谱饼的适合线速范围也较色谱柱宽得多。据此,用装填有HIC填料的色谱饼(10 mm×20 mm i.d.)在12 min内便可完全分离7种标准蛋白。还用装填有HIC填料的色谱饼对重组人粒细胞集落刺激因子(rhG-CSF)进行了复性并同时纯化,在50 min内,仅用一步色谱法就可获得纯度≥97%的rhG-CSF,其质量回收率为39%,比活＞1×108 IU/mg。可以预计,装填极细颗粒的刚性色谱填料的色谱饼可在高负荷条件下进行整体蛋白的高速和高分离度的分离、纯化并同时复性,达到“三高”。     

壳聚糖衍生物吸附剂在蛋白质分离纯化中的应用

综述了近年来甲壳素及壳聚糖衍生物吸附剂、色谱填料、分离膜及医用免疫吸附剂在蛋白质分离纯化中的应用．引用文献65篇．     

高亲水性强阳离子交换色谱填料的制备及其在蛋白质分析中的应用

以具有双孔结构的聚甲基丙烯酸环氧丙酯(PGMA)微球为基质,以葡萄糖进行表面亲水改性,制备了强阳离子交换色谱填料,并将其用于复杂生命体系中生物大分子的快速而高效的分离、分析与纯化。葡萄糖亲水改性增进了填料的生物相容性,提高了蛋白质样品的回收率;双孔结构及较高的比表面积赋予填料良好的柱渗透性和样品负载量。以标准蛋白质为样品,考察了该填料对生物样品的分离性能。以100 mm×4.6 mm的色谱柱分离4种蛋白质,在6 min内实现了基线分离;以溶菌酶为样品,填料的吸附容量为39.5 g/L,在蛋白质快速分离纯化分析中显示了良好的应用前景。     

疏水色谱法的进展及其在生化研究中的应用

 着重评述了疏水色谱法的理论研究及疏水固定相中无机填料、有机填料、非多孔填料以及大孔膜的新发展，并对疏水色谱法在生物大分子的分离、纯化和生化研究中的应用，包括在蛋白质复性、折叠和分子构象变化等方面的应用作了介绍，全文包括６２篇文献和一张表格。     

单分散大孔亲水弱阳离子交换填料的制备及其对鱼精蛋白的分离纯化

以自制的5.0 μm单分散大孔亲水交联聚甲基丙烯酸环氧丙酯(PGMA/EDMA)微球为基质,对其表面进行化学改性,合成弱阳离子交换色谱填料(WCX)。详细考察了该填料对标准蛋白质的分离性能、表面亲水性能、稳定性和重现性以及流速对蛋白保留的影响。实验结果表明,该色谱填料对蛋白的分离性能、重现性及稳定性良好;在流速为3 mL/min时,采用线性梯度洗脱,6 min内可分离4种标准碱性蛋白质,以溶菌酶测定的该填料的动力学吸附容量为29.86 mg/g。将其用于鱼精蛋白的分离纯化,经反相高效液相色谱测定纯化后鱼精蛋白的纯度为99.2%;与商品Shodex IEC SP-825强阳离子交换色谱柱比较,纯化结果几乎一样。     

亚油酸甲酯硅胶聚合物键合相的制备及对蛋白质的分离

1　引　　言从现代分离科学理论计算得出 ,色谱和电泳是目前所知道最好的两种分离方法 ,但是 ,因受各种因素的限制 ,电泳目前尚不能用于生产规模的生物大分子的分离和纯化。这就是把分离和纯化生物大分子 (包括蛋白质、酶、核酸、多糖等 )的研究重点放在色谱上的原因。在生物技术制取蛋白质的多级纯化过程中 ,液相色谱是一个必需步骤。为了获得生物大分子的快速分离就得从基质到键合基团不断改进色谱柱填料。在本文中介绍的柱填料是键合在大孔硅胶上的乙烯基与亚油酸甲酯和二乙烯基苯共聚形成的。从蛋白质混合样品洗脱曲线看出这一填料具有…     

谷胱甘肽键合柱对蛋白的选择性研究

通过将谷胱甘肽键合到硅胶表面合成了同时具有弱阳离子交换(WCX)、疏水(HIC)和氢键作用的多功能色谱填料,该固定相在HIC和WCX模式下对蛋白都有很好的分离效果.实验通过计量置换保留模型对蛋白在谷胱甘肽键合柱上的色谱保留行为及机理进行了研究,结果发现,在流动相盐浓度较低时蛋白根据自身等电点高低通过静电作用力得以分离,而在高盐浓度下疏水和氢键作用力共同决定蛋白的保留.这种多作用力保留模式可有效提高色谱柱的选择性,尤其为蛋白质、多肽及氨基酸的高效分离提供新的解决思路.     

Preparation of hydrophobic interaction chromatographic packings based on monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their application

The monodisperse, poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads with macroporous in the range of 8.0-12.0 microm were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury instrusion method. Based on this media, a hydrophobic interaction chromatographic (HIC) stationary phase for HPLC was synthesized by a new chemically modified method. The prepared resin has advantages for biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The dynamic protein loading capacity of the synthesized HIC packings was 40.0 mg/ml. Six proteins were fast separated in less than 8.0 min using the synthesized HIC stationary phase. The HIC resin was firstly used for the purification and simultaneous renaturation of recombinant human interferon-gamma (rhIFN-gamma) in the extract solution containing 7.0 mol/l guanidine hydrochloride with only one step. The purity and specific bioactivity of the purified of rhIFN-gamma was found more than 95% and 1.3 x 10(8) IU/mg, respectively.     

疏水层析蛋白质动力学与平衡过程的考察

疏水层析是分离生物大分子的常用技术之一，但对疏水层析中蛋白质吸附动力学和平衡过程的研究并不多见．本文对蛋白质疏水吸附动力学和平衡过程作了基本假设，并用实验进行了验证。制备了两种不同丁基密度的疏水琼脂糖介质，用其吸附牛血清白蛋白(BSA)以验证对疏水吸附动力学与平衡过程作的假设，考察了盐浓度及配基密度对蛋白质疏水吸附的影响．还对三种疏水性不同的蛋白质：核糖核酸酶、卵清蛋白和牛血清白蛋白的混合体系进行了分离性能的研究，获得了满意的分离效果．实验表明，蛋白质在疏水介质上的吸附动力学和平衡过程与所作假设相符，在实验条件下等温吸附线符合Langmuir吸附等温方程：研制的丁基琼脂糖疏水介质具有优良的使用性能。     

High-throughput protein precipitation and hydrophobic interaction chromatography: salt effects and thermodynamic interrelation

Salt-induced protein precipitation and hydrophobic interaction chromatography (HIC) are two widely used methods for protein purification. In this study, salt effects in protein precipitation and HIC were investigated for a broad combination of proteins, salts and HIC resins. Interrelation between the critical thermodynamic salting out parameters in both techniques was equally investigated. Protein precipitation data were obtained by a high-throughput technique employing 96-well microtitre plates and robotic liquid handling technology. For the same protein-salt combinations, isocratic HIC experiments were performed using two or three different commercially available stationary phases-Phenyl Sepharose low sub, Butyl Sepharose and Resource Phenyl. In general, similar salt effects and deviations from the lyotropic series were observed in both separation methods, for example, the reverse Hofmeister effect reported for lysozyme below its isoelectric point and at low salt concentrations. The salting out constant could be expressed in terms of the preferential interaction parameter in protein precipitation, showing that the former is, in effect, the net result of preferential interaction of a protein with water molecules and salt ions in its vicinity. However, no general quantitative interrelation was found between salting out parameters or the number of released water molecules in protein precipitation and HIC. In other words, protein solubility and HIC retention factor could not be quantitatively interrelated, although for some proteins, regular trends were observed across the different resins and salt types.     

Effect of surface hydrophobicity distribution on retention of ribonucleases in hydrophobic interaction chromatography

The effect of surface hydrophobicity distribution of proteins on retention in hydrophobic interaction chromatography (HIC) was investigated. Average surface hydrophobicity as well as hydrophobic contact area between protein and matrix were estimated using a classical thermodynamic model. The applicability of the model to predict protein retention in HIC was investigated on ribonucleases with similar average surface hydrophobicity but different surface hydrophobicity distribution. It was shown experimentally that surface hydrophobicity distribution could have an important effect on protein retention in HIC. The parameter "hydrophobic contact area," which comes from the thermodynamic model, was able to represent well the protein retention in HIC with salt gradient elution. Location and size of the hydrophobic patches can therefore have an important effect on protein retention in HIC, and the hydrophobic contact area adequately describes this.     

Novel in situ polymerized coatings for hydrophobic interaction chromatography media

Hydrophobic interaction chromatography (HIC) and other capture media are typically produced by grafting different ligands to base matrices at defined surface densities. This often complicates media production. An alternative approach to media involving in situ radical initiated polymerization was used to graft polymer coatings directly at Sepharose(R) polymeric base matrices. This method appears suitable for producing many different chromatography media on a variety of base matrices. In the present study, it also favorably increased the solution pressure-flow properties of a Sepharose base matrix used to produce HIC media. A wide range of HIC media could be produced by simply varying the reaction ratio of butyl vinyl ether, and hydroxybutyl vinyl ether. The new HIC media was evaluated using five test proteins (bovine serum albumin, ribonuclease A, alpha-chymotrypsinogen A, myoglobin and alpha-lactalbumin). The media exhibited classic HIC behavior, predictably controlled hydrophobicity, plus good protein selectivity, capacity (70mgprotein/ml gel) and often total protein recovery. By modifying the degree of matrix hydrophobicity, we could also reduce effects of protein denaturation often seen with conventional HIC and observed as multiple peaks in the chromatograms. Separation of crude protein extracts from Eschericha coli, expressing a green fluorescent protein (GFPuv) and, a more hydrophobic, recombinantly-modified, tyrosine-tagged green fluorescent protein (YPYPY-GFPuv), was also performed. These proteins were very stable, exhibited significantly different retention times, and could be used to study the ability of the media to work with complex protein mixtures. Such GFP mutants appear ideal for characterizing the performance of chromatographic media.     

Characterization and modeling of nonlinear hydrophobic interaction chromatographic systems

A general rate model was employed in concert with a preferential interaction quadratic adsorption isotherm for the characterization of HIC resins and the prediction of solute behavior in these separation systems. The results indicate that both pore and surface diffusion play an important role in protein transport in HIC resins. The simulated and experimental solute profiles were compared for two model proteins, lysozyme and lectin, for both displacement and gradient modes of chromatography. Our results indicate that a modeling approach using the generate rate model and preferential interaction isotherm can accurately predict the shock layer response in both gradient and displacement chromatography in HIC systems. While pore and surface diffusion played a major role and were limiting steps for proteins, surface diffusion was seen to play less of a role for the displacer. The results demonstrate that this modeling approach can be employed to describe the behavior of these non-linear HIC systems, which may have implications for the development of more efficient preparative HIC separations.     

Microcalorimetric determination of displacement adsorption enthalpies of protein refolding on a moderately hydrophobic surface at 308 K

Both microcalorimetric determination of displacement adsorption enthalpies ΔH and measurement of adsorbed amounts of guanidine – denatured lysozyme (Lys) refolding on the surface of hydrophobic interaction chromatography (HIC) packings at 308 K were carried out and compared with that at 298 K. Study shows that both temperature and concentration of guanidine hydrochloride (GuHCl) affect the molecular mechanism of hydrophobic interaction of protein with adsorbent based on the analysis of dividing ΔH values into three kinds of enthalpy fractions. The adsorption in higher concentrations of GuHCl (>1.3 mol L^–1) at 308 K is an enthalpy-driving process, and the adsorption under other GuHCl concentrations is an entropy-driving process. The fact that the Lys denatured by 1.8 mol L^–1 GuHCl forms a relatively stable intermediate state under the studied conditions will not be changed by temperature.