Boronic Acids as Ligands for Affinity Chromatography

A review on the principles and applications of boronic acids as affinity ligands for the chromatographic separation of carbohydrates,nucleic acid components,glycoproteins,and other small biomolecules.The mechanisms of interactions between boronate ligands and analytes are described.Various boronate ligands and supports are discussed.Examples of the use of boronate affinity chromatography for separation of each class of analytes are presented.

Quantitative analysis of hepatoma-specific α-fetoprotein(HS-AFP) by a new mini-column affinity chromatography and its clinical value in diagnosis of hepatocellular carcinoma

Objective:To establish a convenient and economic method to determine hepatoma-specific α-fetoprotein(HS-AFP) for diagnosis of hepatocellular carcinoma(HCC). Methods:HS-AFP from serum of HCC patients was separated by a mini-column Lens culinaris agglutinin(LCA)-affinity chromatography. The levels of serum total AFP and separated HS-AFP were detected by radioimmunoassay(RIA). Results:Circulating AFP was separated into three peaks(AFP-1,AFP-2,and AFP-3) by LCA-affinity chromatography. During the elution course,the AFP-1 and AFP-2 could be eluted with TE buffer. HS-AFP(AFP-3) from sera of HCC patients was eluted clearly on the LCA-sepharose gel mini-column with a solution containing α-methyl-D-mannoside. It was a part of total AFP and only found in sera of HCC patients. A ratio of more than 15% for HS-AFP to total AFP in serum was considered as a specific marker for HCC diagnosis with higher sensitivity(92.7%) and specificity(88.2%). Conclusion:The new assay for circulating HS-AFP analysis is more sensitive,repeatable,and convenient. Its clini-cal application would be useful to early diagnosis of HCC.

AFFINITY CHROMATOGRAPHY PURIFICATION OF UROKINASE WITH EPICHLOROHYDRIN ACTIVATED AGAROSE MATRIX

1 INTRODUCTIONIn literature,most matrices of affinity chromatography for urokinase(EC 3.4.99.26)purification were prepared by cyanogen bromide activation.However,theseadsorbents usually suffered from the drawback of leakage of the ligand,particularly inalkaline medium,because of the instability of the isourea linkage between the ligandand the spacer or agarose.Moreover,the positively charged imido group of theN-substituted isourea derivative and the hydrophobicity of the spacers might promotenonspecific adsorption.On the contrary,the adsorbents prepared by the method

Isolation of Human Antibodies Against Hepatitis E Virus From Phage Display Library by Immobilized Metal Affinity Chromatography

Objective To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography （1MAC）. Methods Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis. Results His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically. Conclusion Two specific human phage antibodies against hepatitis E virus （HEV） from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.

High Performance Affinity Chromatography of Antithrombin III Based on Monodisperse Poly (glycidyl methacrylate) Beads

A new approach for the separation of antithrombin III with high performance affinity chromatography (HPAC) was described. A novel monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) beads (PGMA) were used as the affinity support. With the water-soluble carbodiimide, heparin was linked covalently to amino-PGMA-beads, which was prepared by amination of PGMA. The adsorbent obtained exhibits high binding activity to antithrombin III (ATIII), good resolution and excellent mechanical properties and can be used under high flow rate.

Separation of Proteins by Electrophoretic Affinity Chromatography

A new kind of electrophoretic affinity chromatography (EAC) for bioseparation was proposed,Separation by EAC was conducted in a multicompartment electrolyzer in which the affinity gel media were packed in one of the central compartments.The presence of an electric field accelerated the migration of proteins inside the gel matrix during adsorption and descrption processes,This led to the increase of the overall speed of separation,The present study was focused on the effect of the strength of the electric field on adsorption and desorption processes.

SYNTHESIS OF INTERFERON-α_A MONOCLONAL ANTIBODY PACKING MATERIAL IN HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PURIFICATION OF RECOMBINANT HUMAN INTERFERON-α_A

A new way for the synthesis of human interferon—α;monoclonal antibody （IFN-α;-McAb） bound to silica gel packing material in high-performance affinity chromatography （HPAFC） has been developed. The high coupling efficiency and specific activity of IFN—α;-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α;（rIFN-α;） rose up to 1.03×10;IU/mg protein and the purification efficiency is appoximately 100 times.

Using frontal affinity chromatography to study how silver binds with particulates

Frontal affinity chromatography was applied to characterizing the mechanism of binding of silver with sediment particulates collected from Lake Ontario, Canada. The results showed that there was one major binding site for Ag+ in the particulates. The binding capacity ranges from 6.06 to 1.01 μ·mol·g-1, and the binding constant (lgK) from 6.23 to 7.43 M-1 in 0.005 M ion strength at pH=3-7. The binding capacity and affinity constant were found to be pH-dependent. It is suggested that the particulate surface site where silver was bound was the anionic base. This study would be helpful for better understanding of the fundamental environmental chemistry of silver in sediments.

Affinity Purification of Insulin by Peptide-Ligand Affinity Chromatography

The affinity heptapeptide(HWWWPAS)for insulin,selected from phage display library,was coupled to EAH Sepharose 4B gel and packed to a 1-mL column.The column was used for the affinity purification of insulin from protein mixture and commercial insulin preparation.It was observed that the minor impurity in the commercial insulin was removed by the affinity chromatography.Nearly 40 mg of insulin could be purified with the 1-mL affinity column.The results revealed the high specificity and capacity of the affinity column for insulin purification.Moreover,based on the analysis of the amino acids in the peptide sequence,shorter peptides were designed and synthesized for insulin chromatography.As a result,HWWPS was found to be a good alternative to HWWWPAS,while the other two peptides with three or four amino acids showed weak affinity for insulin.The results indicated that the peptide sequence of HWWWPAS was quite conservative for specific binding of insulin.

PREPARATION OF CHITOSAN COATED METAL AFFINITY CHROMATOGRAPHY ADSORBENT

A new and an inexpensive adsorbent of chitosan coated silica for immobilized metal affinity chromatography (IMIC) was studied. After a double coating, the chitosan coated on silica beads could be up to 53. 4 mg/g silica beads.When pH＞3. 8, the metal ligand Cu2+ was chelated on the coated chitosan witha bound capacity of 14. 6 mg/g chitosan without introducing iminodiacetic acid(IDA).

IMMOBILIZED CIBACRON BLUE F3G-A ON CROSSLINKED POLY(VINYL ALCOHOL) FOR AFFINITY CHROMATOGRAPHY

Immobilized triazine dye affinity chromatography has been widely used for protein purification. In this paper, Cibacron Blue F3G-A was immobilized, through a spacer arm, onto a rigid hydrophilic porous polymer by reacting an epoxy-group-containing poly(vinyl alcohol) with 6-aminohexyl-N(-Cibacron Blue F3G-A, which was obtained by reacting Cibacron Blue F3G-A with excess of 1,6-diaminohexane, in a pH 8.6 buffer. The epoxy-group-containing poly(vinyl alcohol) was prepared by treating macroporous poly(vinyl alcohol) with excess epichlorohydrin in the presence of NaOH in dimethyl sulfoxide. The macroporous poly(vinyl alcohol) was prepared by hydrolysis of macroporous crosslinked poly(vinyl acetate), which was synthesized by suspension copolymerization of vinyl acetate and triallyl isocyanurate in the presence of butyl acetate and n-heptane as diluents. The Cibacron Blue F3G-A-immobilized poly(vinyl alcohol) was packed in a stainless steel column (250×5 mm I. D.) and the chromatographic behaviors of several proteins (cytochrome c, lysozyme, bovine serum albumin, insulin, and lactate dehydrogenase) were determined.

Adsorption and Step Elution of Urokinase Using Affinity Chromatography-Comparison of Data with Rate Model Simulation

A non-equilibrium chromatographic rate model was employed to simulate the affinity chromatography of urokinase. The chromatography process was developed to a yield of high purity product of urokinase from crude materials. The affinity gel used in the process was prepared by an epichlorohydrin-activation method using epichlorohydrin activated Sepharose 4B as a matrix and p-aminobenzamidine as a ligand. The chromatographic process were numerically simulated and analyzed with the aid of VERSE-LC computer simulator. Considering the basic principles, rate model with the back mixing in column inlet was utilized in simulating and studying the effect of the column inlet pattern on other parameters. Comparison of the simulation results with the experimental data showed that the rate model can be used to describe the affinity chromatography of urokinase in a fixed bed column with satisfactory accuracy.

Separation and Purification of Thrombin-like Enzymes by Affinity Adsorbents

An affinity adsorbent, benzamidine Sepharose 4B, was used to separate and purify thrombin like enzymes. The p aminobenzamidine as a specific ligand was coupled to the matrix-Sepharose 4B. The recombinant thrombin like enzyme-defibrase was used as a model in order to evaluate the efficiency of this biospecific affinity adsorbent. The homogeneity of the enzyme preparation was comfirmed as one band on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Implications from protein uptake kinetics onto dextran-grafted Sepharose FF coupled with ion exchange and affinity ligands

Our previous studies have reported the presence of "chain delivery" effects of protein adsorption onto ion exchangers with polymer-grafted ion-exchange groups, such as dextran-grafted and poly(ethylenimine)-modified Sepharose gels. However, it is unclear if the "chain delivery" occurs on affinity adsorption with specific interactions. This work is designed to address this issue. A dextran-grafted Sepharose gel was prepared, and then the matrix was modified using diethylaminoethyl, a typical ion-exchange group, or octapeptide(FYCHWQDE), an affinity ligand for human immunoglobulin G(h Ig G) to prepare ion-exchange or affinity adsorbents, respectively.Results of h Ig G adsorption showed that the uptake rate represented by the effective diffusivity of h Ig G onto the dextran-grafted ion exchangers was obviously enhanced by the dextran grafting, indicating the presence of"chain delivery" of the bound proteins on the charged groups on the dextran chains. By contrast, the effective diffusivity of h Ig G changed little as ligand density increased on the dextran-grafted FYCHWQDE adsorbents.Their adsorption capacities decreased and effective diffusivities were not accelerated by the dextran grafting.Thus, this work clarified that grafted dextran could not accelerate h Ig G uptake rate on the affinity resins, or in other words, chain delivery did not occur on the specific interaction-based affinity adsorption.

Studies on the homogeneity between high-and lowaffinity glucocorticoid receptor

The homogeneity between the high-and low-affinity glucocorticoid receptors(GR_H and GR_L)was testified by immunoaffinity chromatography using Mab N250(recognizing the immunogenic domain at the N terminal of GR_H)and Mab BuGR1(recognizing the DNA binding domain of GR_H).The specific binding peak of 0.98μmol/L[~3H]triamcinolone acetonide(TA)was higher than that of 52.50nmol/L[~3H]TA.The re-sult suggests that the antigenic determinants of GR_L are similar to those of GR_H.ThecDNA of rat liver GR_H was introduced into GR_H-and GR_L-negative mouse fibroblast cellline E82.A3 by calcium phosphate coprecipitation.A number of clones which expressGR_H were selected with G418(400μg/ml).The results of radioligand binding assay indicatethat GR_H gene is expressed successfully and GR_L also may be encoded by GR_H gene.

Oligo(dT)亲和层析介质的载量比较和机制分析

针对Oligo(d T)亲和层析介质的吸附性能,以poly(A)为模型分子,考察了4种Oligo(d T)亲和层析介质的静态吸附平衡、吸附动力学和动态结合载量(DBC),探讨了载量影响相关机制。结果表明,4种介质的合适吸附条件均为0.6 mol·L-1Na Cl、p H=6~7;Monomix d T20静态吸附容量最大,且poly(A)能扩散至介质微球深层孔内,而Poros Oligo(d T)25、Praesto Jetted (d T)25和Nano Gel d T20等3种介质中poly(A)均主要为表层吸附、静态吸附容量稍低;对于DBC,Nano Gel d T20和Monomix d T20的10%穿透的DBC较高,而Poros Oligo (d T)25和Praesto Jetted (d T)25相对略低。经分析,影响载量的主要因素包含基质种类、微球孔径、配基密度、间隔臂和配基长度等。对于基质种类,聚苯乙烯基质可能孔道结构较为特别。对于微球孔径,应针对不同大小的m RNA分子定制不同孔径的微球,以平衡传质阻力与可及吸附表面积之间的矛盾,从而增大DBC。

CTA中空纤维亲和色谱的制备及其吸附性能

为探究三醋酸纤维素(CTA)中空纤维亲和色谱的吸附性能,以CTA中空纤维膜为基膜,通过水解、1,4-丁二醇二缩水甘油醚(EGDE)交联活化、接枝1,6-己二胺(HMDA)作为间隔臂对膜表面进行改性,然后以亚氨基二乙酸(IDA)为配基,螯合Cu^(2+)制备了改性CTA中空纤维亲和色谱.对于γ-球蛋白的静态吸附研究表明:当吸附液γ-球蛋白质量浓度为1 mg/mL、离子浓度为0.2 mol/L和pH=8时,有最大静态吸附容量2.4 mg/cm^(3).且其吸附过程符合Langmuir等温吸附模型.对于γ-球蛋白的动态吸附研究表明:在实验研究的范围内,不同流速下的穿透曲线都呈现出类似“S”型,流速并不影响CTA中空纤维亲和色谱上γ-球蛋白的结合能力;γ-球蛋白的动态吸附容量随其初始浓度的增加而增加.

定量磷酸化蛋白质组解析17β-雌二醇致死效应的细胞调控过程

17β-雌二醇(E2)是人体内一种重要的内分泌激素,在生理浓度下(0.2~1.0 nmol/L)对生殖系统、乳腺等靶器官的生长发育起着重要的调节作用。但很多研究表明,高剂量(μmol/L~mmol/L)的E2能够诱导肿瘤组织消退和细胞凋亡,其具体调控机制尚不明确。本工作聚焦于高剂量(μmol/L)的E2致死效应,首先分析了μmol/L水平的E2对HeLa细胞表型的影响,发现在1~10μmol/L下E2以浓度依赖的形式抑制HeLa细胞增殖,并诱导HeLa细胞发生死亡,其中,用5μmol/L E2处理2天后可使约74%的HeLa细胞增殖受到抑制,并引起约50%的HeLa细胞死亡。在此基础上,为了探究高剂量E2诱导细胞死亡的内在调控过程,将基于固相萃取(SPE)的固定化钛离子亲和色谱技术(Ti 4+-IMAC)与基于数据非依赖采集模式(DIA)的蛋白质组定量技术结合,用于筛选HeLa细胞内参与高剂量(μmol/L)E2致死效应调控过程的磷酸化位点。最终,在5μmol/L E2和二甲基亚砜(DMSO)处理的HeLa细胞中共鉴定到超过10000个磷酸化位点;t检验分析发现,在E2处理后,有924个磷酸化位点(对应599个蛋白质)的丰度发生了显著变化(显著性水平(p)<0.01,|log 2(倍数变化)|≥1),推测其可能参与调控E2致死效应过程。此外,有453个磷酸化位点(对应325个蛋白质)仅单独发生在E2或DMSO处理后的HeLa细胞样品中,表明这些磷酸化位点在E2处理后发生了磷酸化或去磷酸化,也可能参与E2致死效应的调控过程。分别对以上两种方式筛选的E2调控的磷酸化蛋白质进行富集分析,发现这些磷酸化蛋白质主要参与细胞分裂、核糖体/核质转运、信使核糖核酸(mRNA)加工/剪接及转录等过程,表明高剂量的E2可能通过调控核糖体及mRNA加工等过程影响蛋白质转录,进而诱导细胞发生死亡。此外,我们发现表皮生长因子受体(EGFR)和丝裂原活化蛋白激酶(MAPK)家族蛋白(包括MAPK1、MAPK4和MAPK14)上多个磷酸化位点的修饰水平在高剂量E2处理后发生了明显变化,表明EGFR和MAPK信号通路可能在雌激素诱导的细胞死亡中起着重要调控作用。本实验得到的磷酸化蛋白质组定量结果有助于进一步了解高剂量E2的内在调控过程,为后续解析高剂量E2的作用机制及疾病的治疗提供了参考。

色谱技术在抗体分离纯化中的应用进展

抗体作为一类重要的生物药物,在疾病诊断和治疗等方面拥有广阔的前景。抗体药物的需求逐年上升,生产规模不断扩大,下游抗体纯化已经成为抗体药物生产的瓶颈。色谱技术具有选择性好、分离效率高等优点,在抗体纯化领域占据主导地位。抗体分离纯化流程一般包括样品的粗提、粗提物精制和抗体精纯3个步骤,每个步骤都涉及色谱分离技术。目前已有多种色谱技术(如亲和色谱、离子交换色谱、疏水作用色谱、混合模式色谱以及新型的温敏色谱等)应用于抗体的分离纯化。本文介绍了近年来国内外各种色谱技术在抗体分离纯化方面所取得的研究进展,并对抗体色谱分离技术的未来发展趋势进行了展望。