Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography

This article has proposed an artificial chaperone-assisted immobilized metal affinity chromatography (AC-IMAC) for on-column refolding and purification of histidine-tagged proteins. Hexahistidine-tagged enhanced green fluorescent protein (EGFP) was overexpressed in Escherichia coli, and refolded and purified from urea-solubilized inclusion bodies by the strategy. The artificial chaperone system was composed of cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD). In the refolding process, denatured protein was mixed with CTAB to form a protein–CTAB complex. The mixture was then loaded to IMAC column and the complex was bound via metal chelating to the histidine tag. This was followed by washing with a refolding buffer containing β-CD that removed CTAB from the bound protein and initiated on-column refolding. The effect of the washing time (i.e., on-column refolding time) on mass and fluorescence recoveries was examined. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC led to 99% pure EGFP with a fluorescence recovery of 80%. By comparison at a similar final EGFP concentration (0.6–0.8 mg/mL), this fluorescence recovery value was not only much higher than direct dilution (14%) and AC-assisted refolding (26%) in bulk solutions, but also superior to its partner, IMAC (60%). The operating conditions would be further optimized to improve the refolding efficiency.     

Separation of polythiolate peptides and metallopeptides by covalent affinity chromatography

Summary Metallothionein (MT) a low-molecular weight polythiolate metallopeptide was isolated quantitatively by covalent affinity chromatography on Sepharose DTNB support of our own synthesis. The protein was prepared from the vital organs of rats exposed to heavy metals (Hg, Cd).Isolation of low-molecular weight polythiolate and thiolate proteins of a small number of SH-groups are reported. Changes in II and III order structure of the protein and aggregation resulting from chaotic formation of disulphide bridges were observed.A mechanism of separation of polythiolate proteins by covalent chromatography, based on the presented data, is suggested.Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

Evaluation and optimization of the metal‐binding properties of a complex ligand for immobilized metal affinity chromatography

The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu²⁺ to l ‐glutamic acid were measured, the metal ion‐binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal‐binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum‐binding conditions of Cu²⁺ on the l ‐glutamic acid‐silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal‐binding behaviors.     

增强型绿色荧光蛋白的色谱分离和纯化

增强型绿色荧光蛋白(EGFP)是生物领域常用的标记物。在前期成功克隆表达EGFP的基础上,本实验建立了两步分离纯化EGFP的色谱方法,并验证其分离纯化效果,检验EGFP的活性。首先用金属螯合亲和色谱柱HisTrap HP对EGFP的重组菌体破碎上清液进行初步分离,再用葡聚糖凝胶排阻色谱柱Sephadex G-10 HR对其进行脱盐纯化。采用丙烯葡聚糖凝胶排阻色谱柱Sephacryl S-300 HR和十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测分离纯化后的EGFP纯度。最后通过荧光分光检测器和非变性聚丙烯酰胺凝胶电泳(Native-PAGE)验证分离纯化后的EGFP是否具有荧光活性。结果表明该方法可以简便快速地分离纯化EGFP,纯度超过98%,同时保持了EGFP的荧光活性。     

一种凝血酶亲和色谱介质的制备方法

对凝血酶-琼脂糖亲和色谱介质的制备方法进行了研究。首先使用凝血酶和溴化氰活化的琼脂糖制备凝血酶-琼脂糖亲和色谱介质,然后用生色底物法考察亲和色谱介质上凝血酶的活性,以凝血酶活性为指标对最佳偶联条件进行了优化。结果表明最佳条件为使用pH 8.3的Na₂CO₃-NaHCO₃溶液(含0.5 mol/L NaCl)为缓冲溶液,凝血酶用量为每1 g色谱介质加入凝血酶200 U,室温反应10 h。在最佳条件下所制备的色谱介质有较好的稳定性,在4℃条件下存放40天,亲和介质上的凝血酶活性仍有70.6%保留。该亲和色谱介质可广泛用于含凝血酶抑制剂的天然药物筛选和分离纯化。     

Purification of neutral protease by dye affinity chromatography

Twenty triazinic dyes were assayed as ligands for the chromatographic affinity purification of a neutral protease from Flavourzyme^™, a commercial preparation. Screening at pH 4.0 allowed the selection of eight dyes on the basis of their high protease adsorption. When the pH was set to 5.0 in order to increase selectivity, only Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A maintained protease adsorption at high values. Neither maximum capacities nor dissociation constants calculated from isotherms measured at 8 and 25°C showed great differences. By contrast, a strong temperature effect was evidenced in the elution step: elution at 8°C allowed 70, 81, and 98% recovery of adsorbed protease with Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A, respectively, whereas only 20% recovery was attained at 25°C. Based on the results obtained, a purification process for the neutral protease contained in Flavourzyme with Cibacron Blue F3G-A as the affinity ligand was developed, yielding 96% of electrophoretically pure enzyme in a single step, the specific activity rising from 850 to 3650 U/mg.     

Activation of trisacryl gels with chloroformates and their use for affinity chromatography and protein immobilization

In this study we describe the activation with chloroformates of Trisacryl-GF-2000, a new synthetic gel support that is stable, hydrophilic, and contains large amounts of hydroxyl groups available for activation. Of all the reagents tested, the activation withN-hydroxysuccinimide-chloroformate andp-nitrophenylchloroformate in organic solvents provides the best activation yield and subsequent coupling. When Trisacryl was activated in acetone with the chloroformates in the presence of 4-dimethylaminopyridine as base and catalyst, up to 30% of the hydroxyl groups, (i.e., 1/repeating unit) could be activated. Amino-containing ligands and proteins could be coupled to these carriers at pH 8 or higher. For better results in affinitychromatographic applications, spacers of ε-amino caproic acid or diaminohexane were introduced. The efficacy of these columns was demonstrated by purification of enzymes, antibodies, and antigens. The performance of these new columns were compared with that of Sepharose columns activated in various ways. In every case, the properties of the Trisacryl support proved superior with particular reference to the purity of the product obtained.     

A preliminary study for isolation of catalytic antibodies by histidine ligand affinity chromatography as an alternative to conventional protein A/G methods

Catalytic autoimmune antibodies from the sera of lupus patients were purified using histidyl-aminohexyl-Sepharose gel and compared with the antibodies purified with protein A and protein G affinity chromatography. The IgG preparations from the histidine affinity column had a much higher catalytic activity in hydrolyzing the peptide substrate Pro-Phe-Arg-methyl-coumarinamide compared to the antibodies obtained by the conventional protein A/G method. This preservation of catalytic activity is attributed to the gentle buffer conditions used in the histidine ligand method that allowed the integrity of three-dimensional structure of purified catalytic antibodies. Thus, histidine affinity offer a superior method for isolating autoimmune catalytic antibodies.     

亲和分离在蛋白质泛素化修饰研究中的应用进展

蛋白质泛素化是真核生物最普遍、最复杂的翻译后修饰方式之一,在细胞的信号转导、生长、发育、代谢等生命过程中发挥着重要作用。泛素化过程的失调则与神经退行性疾病、炎症反应、癌症等重大疾病的发生发展密切相关。分析和研究蛋白质泛素化的结构与功能,可望为认识生命、探索疾病调控内在规律和发现新的诊断策略提供重要信息。生命体系的高度复杂性,泛素化修饰位点、结构类型的多变和多样性,时空动态变化等特点给蛋白质泛素化分析研究带来了巨大的挑战。亲和分离以其高选择性成为泛素化蛋白质结构与功能研究的有力工具。免疫亲和分离法基于抗原-抗体相互作用,是最为经典的分离分析方法,已广泛应用于泛素化蛋白质或肽段的富集分离。源于天然泛素受体的泛素结合结构域(ubiquitin binding domains, UBDs)可与泛素或多聚泛素链相互作用。UBDs和基于此发展起来的串联泛素结合实体(tandem ubiquitin-binding entities, TUBEs)已成为蛋白质泛素化功能研究的热门识别分子。各种多肽类化合物的发展也为蛋白质泛素化的结构和功能解析提供新工具。此外,多种亲和识别配基的联合使用,在蛋白质泛素化修饰的高特异性、高灵敏度分析中展现了独特的优势,为认识生命体内的泛素化修饰提供了重要保障。该文对亲和分离方法在蛋白质泛素化修饰分析中的应用及进展进行了综述。     

Foot and mouth disease virus concentration and purification by affinity chromatography

Foot and mouth disease virus, (FMDV) from a crude cell lysate was purified in a single step by affinity chromatography with heparin as a ligand. The virus eluted from an Heparin-Ultrogel A₄R column at 1M sodium chloride in 10 mM sodium phosphate buffer, pH 7.0, while most cell protein and albumin did so at lower concentrations of sodium chloride in the same buffer. Purity of the eluted fraction containing the virus was assessed by SDS-PAGE, HPLC, ultracentrifugation, and UV absorption spectrum. With this method, intact viral particles are recovered in high yield (over 90%) and specific virus purity increases nearly 1000-fold. The capacity of the Chromatographic matrix for the virus was found to be 1.1 mg viral mass per mL of hydrated gel.     

Antibody purification by affinity chromatography based on small molecule affinity ligands identified by SPR-based screening of chemical microarrays

Libraries of small molecules were searched for Fc-fragment selective binders to a recombinant human antibody ("MDJ8″, IgG(1)-subtype, κ-light chain) via SPR-based screening of chemical microarrays. Identified hit structures were immobilised on NHS-activated Sepharose for the determination of MDJ8 binding and selectivity versus typical proteineous impurities represented by the spend cell culture supernatant. Columns were packed and the most promising ligands further characterized in terms of binding constants, binding kinetics, as well as dynamic and equilibrium binding capacities. The performance of the best ligand, 2A10, was compared to standard Protein A chromatography. Using ligand 2A10 antibody capture from unprocessed cell culture supernatants was possible at similar recovery yield (>90%), purity (>80%), and eluting concentration (approximately 1 g/L) as with Protein A. Affinity constants (K(d)) of 2A10 were an order of magnitude higher than for the Protein A material, but still in the nM-range, while maximum binding capacities and binding kinetics were in the same order of magnitude. Ligand 2A10 was also able to capture a murine monoclonal antibody, again with similar efficiency as Protein A, as well as a number of humanised therapeutic antibodies. Antibody elution from the 2A10 column was possible using the Protein A standard protocol, i.e. 100mM glycine HCl pH 3.0, but also at near physiological pH, when some organic solvent or modifiers were present. Ligand 2A10 thus constitutes a cheaper, more robust alternative to Protein A as possible generic antibody binder. Moreover, the outlined approach to ligand selection could in principle by used to create suitable affinity ligands for other high value biotech products.     

Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation

Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation have been prepared. They were obtained by a “ post-magnetization” procedure involving a simple treatment of the various affinity gels with magnetic ferrofluid. The magnetic biospecific adsorbents tested include magnetic protein A-Sepharose for isolation of IgG antibodies, magnetic human serum albumin (HSA)-Sepharose for anti-HSA isolation, and magnetic 2′,5′-ADP for isolation of glucose-6-phosphate dehydrogenase from baker’s yeast and hemolyzates of human red blood cells. For the latter enzyme, a 11,000-fold purification was achieved in one step.     

Understanding ligand–protein interactions in affinity membrane chromatography for antibody purification

Affinity chromatography with Protein A beads has become the conventional unit operation for the primary capture of monoclonal antibodies. However, Protein A activated supports are expensive and ligand leakage is an issue to be considered. In addition, the limited production capabilities of the chromatographic process drive the research towards feasible alternatives. The use of synthetic ligands as Protein A substitutes has been considered in this work. Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports. The resulting affinity membranes have been experimentally characterized with pure immunoglobulin G (IgG). The effects of the membrane support and of the spacer arm on the ligand–ligate interaction have been studied in detail. Experimental data have been compared with molecular dynamic simulations with the aim of better understanding the interaction mechanisms. Molecular dynamic simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the interactions of surface, spacer and ligand with IgG, contribute to the formation of the bond between protein and affinity membrane.     

Hen ovomucoid-agarose: A new conjugate for the isolation by affinity chromatography of UDP-galactose: Glycoprotein galactosyl-transferase

A new conjugate for the affinity chromatography of UDP-galactose:glycoprotein galactosyltransferase has been synthesized by coupling hen ovomucoid, a ligand similar to the acceptor substrate, to agarose. The hen ovomucoid-Sepharose conjugate binds galactosyl transferase more tightly that other acceptor-Sepharose conjugates. The new adsorbent gives comparable yields and purifications with those obtained by ligands similar to the nucleotide moiety of the substrate and to the “specifier” protein, α-lactalbumin. The soluble galactosyltransferase from rat ventral prostate is effectively removed from the high speed supernatant by an ovomucoid-Sepharose column. The enzyme can be eluted with buffer containing EDTA andN-acetylglucosamine in a high yield (75–80%) and in a purified form (4000-fold purification). The stability of ovomucoid to heat and to high concentrations of urea and its inhibition of some proteases makes the conjugate easy to operate with an quite useful even with rather crude preparations.     

Inhibitors of an AdoMet-dependent 3-amino-3-carboxypropyl transferase and their use as ligands for protein affinity chromatography

The screening of potential affinity ligands and the development of a two-stage affinity purification of an S-adenosylmethionine-dependent 3-amino-3-carboxypropyl transferase involved in the biosynthesis of the β-lactam antibiotic nocardicin A is described from a cell-free extract of Nocardia uniformis subsp. tsuyamanesis. This protein is particularly sensitive to ion exchange and salt effects necessitating a pair of neutral ligands to be used in the final purification.     

亲和色谱法筛选中药中血管紧张素转化酶抑制剂

以壳聚糖微球为载体、戊二醛（glutaraldehyde,GA）为交联剂对血管紧张素转化酶（Angiotensin converting enzyme,ACE）进行固定化．用固化的ACE作为亲和介质,利用血管紧张素转化酶抑制剂（Angiotensin convertingenzym einhibitor,ACEI）与ACE之间的亲和作用,结合高效液相色谱对亲和前后的体系进行检测,比较两者各组分色谱峰的差异,以此实现快速筛选复杂体系中的ACE抑制剂．应用赖诺普利（Lisinopril）、九肽抑制剂、依那普利（Enalapril）、培哚普利（Perindopril）、卡托普利（Captopril）等已上市的ACEI对方法进行验证,反映方法具有高度选择性．将方法应用于中药地龙及山楂筛选,发现共有5个组分与ACE有亲和作用,并且都能抑制ACE酶活性,它们对酶活性抑制的IC50值在0．45～4．62μg／mL范围．通过对亲和方法重现性考察,6次测定的相对标准偏差小于1％,说明方法可靠．提出的亲和色谱．色谱指纹差异法非常适合于从中药及天然产物等复杂混合物库中快速筛选靶点活性物质．     

混合抗体的亲和色谱分离策略

乳腺生物反应器可以高效表达重组人单克隆抗体,但是目标产品与乳液原料中的牛抗体性质、结构非常类似,分离难度很大。本文对牛抗体和重组人抗体的种属差异进行了分析,并在此基础上制定了新型分离策略,采取Protein A亲和色谱和免疫亲和色谱来解决混合抗体的分离问题,并讨论了色谱洗脱模式对分离效果的影响。结果表明,Protein A亲和色谱结合梯度洗脱可以有效地纯化得到混合抗体,但是难以彻底分离重组人抗体和牛抗体;相比之下,使用Protein A亲和色谱结合置换色谱模式可以更加高效地分离混合抗体,最终可以得到纯度高达95%以上的重组人抗体,回收率可达95%以上。免疫亲和色谱同样可以有效地分离纯化重组单克隆抗体,且其通用性更强,可以应用于任何动物乳腺表达重组人抗体的分离纯化中。     

The isolation and purification of pre-S2 containing hepatitis B virus surface antigen by chemical affinity chromatography

A simple, rapid, and efficient method was developed to isolate and purify pre-S2 containing HBsAgs from the plasma of a single chronic carrier of HBsAg (adw) by ammonium sulfate fractionation, hydroxyapatite column chromatography, and polymerized human serum albumin-affinity column chromatography. About 500 μg of pre-S2 containing HBsAg was obtained from 140 mL of plasma containing 4,200 μg of HBsAg. Two purified pre-S2 containing HBsAgs were analyzed by SDS-polyacrylamide gel electrophoresis and their molecular weights were determined to be 31,000 and 68,000 respectively. No significant amount of HBsAg or its derivative was detected in the final product.