复合配基亲和色谱填料的制备
|
摘要
亲和色谱是一种高效简便的蛋白纯化分离技术，高效亲和色谱(HPAC)是在经典亲和色谱技术上结合高效液相色谱(HPLC)的特点发展起来的新技术，它具有分离速度快、分离效果好、回收率高和操作简单等优点，适用于批量样品的分析和制备。8微米左右的表面多孔单分散交联聚甲基丙烯酸环氧丙酯／甲基丙烯酸乙二醇酯P(GMA-EDMA)微球可作为高效液相色谱柱填料中的基质材料。本文将P(GMA-EDMA)微球作为高效液相色谱的基质材料，三嗪染料和金属作为亲和配基，制备一种新型的复合配基亲和色谱填料并将其用于对蛋白质的分离纯化，主要做了以下研究工作：
1．以分散聚合法制备的2微米聚苯乙烯微球为种子，GMA、EDMA为单体，用一步种子溶胀法制备了表面多孔的P(GMA-EDMA)微球。该微球7微米左右，粒径单分散，表面多孔，刚性好，适合用做高效色谱柱填料。
2．以自制P(GMA-EDMA)微球为基球，通过不同的连接方式，将染料配基PBMX-R和金属Ni~(2+)固定在基球上，制备成两种复合配基高效亲和色谱填料Ni~(2+)-IDA-PB-P(GMA-EDMA)和Ni~(2+)-PB-P(GMA-EDMA)。
3．用匀浆法将所制的复合配基亲和色谱填料填装成高效亲和色谱柱，通过比较复合配基亲和色谱与单一亲和配基亲和色谱对蛋白质的保留行为和吸附情况，探究复合配基亲和色谱对蛋白质的保留机理，将含有染料配基和金属配基的Ni~(2+)-IDA-PB-P(GMA-EDMA)、Ni~(2+)-PB-P(GMA-EDMA)复合配基亲和色谱柱与含有单一配基的亲和色谱柱对蛋白质的吸附情况进行比较，选择最佳色谱柱。
4．在10×0.46cm的Ni~(2+)-IDA-PB-P(GMA-EDMA)色谱柱上，探讨了pH、流速和固定了不同金属离子的色谱柱对溶菌酶分离的影响，得出溶菌酶的最佳淋洗条件为：流动相A为0.02mol／L pH6.86磷酸盐缓冲液，B为含1.0mol／L NaCl的pH6.86磷酸盐缓冲液，淋洗梯度为15_minB从0到100％，进样量为20μL，流速为0.5_ml／min。在最佳淋洗条件下对不同浓度溶菌酶进行测定，该高效亲和色谱柱具有良好的线性、重现性、稳定性等色谱性能。实际应用中，它能将鸡蛋清样品中的溶菌酶成功分离，并从猪心中分离纯化出高浓度的细胞色素C。
High-performance Affinity Chromatography(HPAC) is a method combined with classical affinity chromatography in an HPLC system. It has advantages of rapid analysis, high column efficiency and high recovery. Affinity chromatographic column based on porous particles of 8μm P(GMA-EDMA) is applicable for the analysis and micropreparative separation of proteins. In this paper, we prepared multiple-ligand affinity resin in which P(GMA-EDMA) microspheres were used as the support, and dye ligand and metal ligand were incorporated into these microbeads for the separation and purification of proteins. The main work follows:
1. Porous particles, P(GMA-EDMA) micropheres with diameter of 7μm were prepared by a single-step swelling and polymerization method, using polystyrene micropheres prepared by dispersion polymation as seed particles and GMA、EDMA as monomers. The synthesized monodisperse P(GMA-EDMA) resin possesses uniform、porous characteristics and high mechanic intensity.
2. Procion blue MX-R and Ni~(2+)were coupled with P(GMA-EDMA) microspheres through different ways, thus two novel multiple-ligand affinity resins Ni~(2+)-IDA-PB-P(GMA-EDMA)、Ni~(2+)-PB-P (GMA-EDMA) were prepared.
3. The multipe-ligand immobilized P(GMA-EDMA) beads were slurry packed into stainless steel column.The protein retention machanism on multiple-ligand affinity chromatography was explored by comparing protein retention behavior and adsorption ability on multiple-ligand affinity chromatography with on mono-ligand affinity chromatography. Ni~(2+)-IDA-PB-P(GMA-EDMA) column was selected as the best column for protein separation and purification.
Ni~(2+)-IDA-PB-P(GMA-EDMA)column(10×0.46cm I.D.) were investigated in selectivity, stabilization, protein loading capacity and recovery et. al., using lysozyme as standard protein. On this column,lysozyme was well eluted under the selected experimental conditions. The eluent flow rate was set at 0.5 ml/min with injection volume of 20μL and the mobile phase linear gradient was set in 15 min from 0 to 1 mol/L sodium chloride in 20 mmol/L phosphate buffer at pH 6.86. The column has low column backpressure, good stability, high protein mass recovery, and was factual used for the separation of lysozyme from egg white and purification of cytochrome C from procine heart.
1．以分散聚合法制备的2微米聚苯乙烯微球为种子，GMA、EDMA为单体，用一步种子溶胀法制备了表面多孔的P(GMA-EDMA)微球。该微球7微米左右，粒径单分散，表面多孔，刚性好，适合用做高效色谱柱填料。
2．以自制P(GMA-EDMA)微球为基球，通过不同的连接方式，将染料配基PBMX-R和金属Ni~(2+)固定在基球上，制备成两种复合配基高效亲和色谱填料Ni~(2+)-IDA-PB-P(GMA-EDMA)和Ni~(2+)-PB-P(GMA-EDMA)。
3．用匀浆法将所制的复合配基亲和色谱填料填装成高效亲和色谱柱，通过比较复合配基亲和色谱与单一亲和配基亲和色谱对蛋白质的保留行为和吸附情况，探究复合配基亲和色谱对蛋白质的保留机理，将含有染料配基和金属配基的Ni~(2+)-IDA-PB-P(GMA-EDMA)、Ni~(2+)-PB-P(GMA-EDMA)复合配基亲和色谱柱与含有单一配基的亲和色谱柱对蛋白质的吸附情况进行比较，选择最佳色谱柱。
4．在10×0.46cm的Ni~(2+)-IDA-PB-P(GMA-EDMA)色谱柱上，探讨了pH、流速和固定了不同金属离子的色谱柱对溶菌酶分离的影响，得出溶菌酶的最佳淋洗条件为：流动相A为0.02mol／L pH6.86磷酸盐缓冲液，B为含1.0mol／L NaCl的pH6.86磷酸盐缓冲液，淋洗梯度为15_minB从0到100％，进样量为20μL，流速为0.5_ml／min。在最佳淋洗条件下对不同浓度溶菌酶进行测定，该高效亲和色谱柱具有良好的线性、重现性、稳定性等色谱性能。实际应用中，它能将鸡蛋清样品中的溶菌酶成功分离，并从猪心中分离纯化出高浓度的细胞色素C。
High-performance Affinity Chromatography(HPAC) is a method combined with classical affinity chromatography in an HPLC system. It has advantages of rapid analysis, high column efficiency and high recovery. Affinity chromatographic column based on porous particles of 8μm P(GMA-EDMA) is applicable for the analysis and micropreparative separation of proteins. In this paper, we prepared multiple-ligand affinity resin in which P(GMA-EDMA) microspheres were used as the support, and dye ligand and metal ligand were incorporated into these microbeads for the separation and purification of proteins. The main work follows:
1. Porous particles, P(GMA-EDMA) micropheres with diameter of 7μm were prepared by a single-step swelling and polymerization method, using polystyrene micropheres prepared by dispersion polymation as seed particles and GMA、EDMA as monomers. The synthesized monodisperse P(GMA-EDMA) resin possesses uniform、porous characteristics and high mechanic intensity.
2. Procion blue MX-R and Ni~(2+)were coupled with P(GMA-EDMA) microspheres through different ways, thus two novel multiple-ligand affinity resins Ni~(2+)-IDA-PB-P(GMA-EDMA)、Ni~(2+)-PB-P (GMA-EDMA) were prepared.
3. The multipe-ligand immobilized P(GMA-EDMA) beads were slurry packed into stainless steel column.The protein retention machanism on multiple-ligand affinity chromatography was explored by comparing protein retention behavior and adsorption ability on multiple-ligand affinity chromatography with on mono-ligand affinity chromatography. Ni~(2+)-IDA-PB-P(GMA-EDMA) column was selected as the best column for protein separation and purification.
Ni~(2+)-IDA-PB-P(GMA-EDMA)column(10×0.46cm I.D.) were investigated in selectivity, stabilization, protein loading capacity and recovery et. al., using lysozyme as standard protein. On this column,lysozyme was well eluted under the selected experimental conditions. The eluent flow rate was set at 0.5 ml/min with injection volume of 20μL and the mobile phase linear gradient was set in 15 min from 0 to 1 mol/L sodium chloride in 20 mmol/L phosphate buffer at pH 6.86. The column has low column backpressure, good stability, high protein mass recovery, and was factual used for the separation of lysozyme from egg white and purification of cytochrome C from procine heart.
引文
[1] Janson J C, Lars Ryden. Protein Purification: Principles, high resolution methods, and application. New York: VCH, 1989. 332
[2] 唐欢欢，王林，亲和色谱在医药生物中的应用，医药化工，2005，(5)：24-29
[3] 韩金玉，那平．亲和色谱纯化蛋白质新进展，色谱，1996，14(6)；447-450
[4] Cuatrecasas P., Wilchek M., Single-step purification of avidine from egg white by affinity chromatography on biocytin-Sepharose columns. Biochem. Biophys. Res. Commun, 1968, 33(2): 235-244
[5] Denizli A., Piskin E. Dye-ligand affinity systems. J. Biochem. Biophys. J. Biochem. Biophys. Methods, 2001, 49: 391-416
[6] 任军，贾凌云，亲和色谱仿生配基的筛选和设计，分析化学，2005，(9)：1345-1349
[7] 周绪斌，宣华，李荣秀，组特异性亲和配基在分离纯化中的应用，中国生化药杂志 2000，21(6)：308-310
[8] Ozkara S, Yacuz H, Denizli A. Purification of immunoglobulin G from human plasma by metal-chelate affinity chromatography, J. Appl. Polym. Sci., 2003, 89 (6): 1567-1572
[9] Lowe C R, Pearson J C. Affinity chromatography on immobilized dyes. Methods Enzymol., 1984, 104: 97-113
[10] Clonis Y D, Labrou N E, Kotsira V, Mazitsos K. Biomimetic dyes as affinity chromatography tools in enzyme purification J. Chromatogr. A, 2000, 891 (1); 33-44
[11] Starkenstein E., Biochem. Z, 1910, 24: 210-218
[12] Cuatrecasas P., Wilchek M., Anfinsen C. B. Selective enzyme purification by affinity chromatography. Proc. Nat. Acad. Sci. U S., 1968, 61(2): 636-643
[13] Livshits M A, Mirzabekov A D. Theoretical analysis of the kinetics of DNA hybridization with gel-immobilized oligonucleotides. Biophys J., 1996, 71(5): 2795-2801
[14] Jamn J.The kinetics of protein-protein recognition. Proteins: Struct Funct Genet, 1997, 28(2): 153-161
[15] Meir Wilchek, Talia Miron, Thirty years of affinity chromatography. Reactive &Funct. polym, 1999, 41: 263-268
[16] Bonnerjea J., Dh s., Hoare M. Protein purification: the right step at the right time. Biotech, 1986, 4: 954-958
[17] Groman E. V., Wilchek M. Recent developments in affinity chromatography supports. Trends Biotech, 1987, 5 (8): 220-224
[18] Tjonnfjord G E, Steen R,Veiby O.P. Lineage commitment of CD34+ human hematopoietic progenitor cells. Exp Hematol, 1996, 24(5): 875—882
[19] Liang X., Lubman D. M., Ross D. T. On-Probe Immunoaffinity Extraction by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Anal Chem, 1998, 70(3): 498-503
[20] Porath J, Carlsson J, Olsson I, Belfrage G. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature, 1975, 258(5536): 598
[21] 李蓉，陈国亮，赵文明．蛋白质在合成阳离子交换剂上的色谱特性研究色谱，2004，22(2)：134-137．
[22] J W Wong, R L Albright, N H L Wang.Immobilized metal ion affinity chromatography(IMAC) chemistry and bioseparation application. Separation and Purification Methods, 1991, 20 (1): 49-106
[23] H Y Reina, A R Manriquez, B R Wong, Selectivity of IMAC Columns in Trypsin Inhibitor Purification. Biotechnol. Prog., 2001, 17(4): 729-733
[24] Pearson, R. G. In: Rearson, R. G., editor. Hard and soft acids and bases. Hutchington &Ross, 1973. p. 53-9, 67-85.
[25] Grigoriy S. Chaga. Twenty-five years of immobilized metal ion affinity chromatography:past,present, and future.J.Biochem.Biophys.Methods 49(2001)313-334
[26] Z. Y. Ma, Y. P. Guan, Synthesis of Magnetic Chelator for High-Capacity Immobilized Metal Affinity Adsorption of Protein by Cerium Initiated Graft Polymerization.Langmuir,2005, 21(15), 6987-6994
[27] Aurelio Hidalgo, Chlorpromazine N-Demethylation by Hydroperoxidawd Activity of Covalent Immobilized Lipoxygenase.Biotechnol. Prog. 2004, 20(5), 1578—1582
[28] Luciana Cristina Lins de Aquino, Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin . J.Chromatogr.B, 2006,834(1): 68-76
[29] MARTIN LUND.Purification and Identification of Water-Soluble Phosphopeptides from Cheese Using Fe(III) Affinity Chromatography and Mass Spectrometry. J. Agric. Food Chem. 2004, 52, 6616-6622
[30] M. Yakup Aryca. Dye-ligand and metal chelate poly(2-hydroxyethylmethacrylate) membranes for affinity separation of proteins. J.Chromatogr. A, 1998,799(1): 83-91
[31] Chun-Yi Wu. Analysis of protein adsorption on regenerated cellulose-based immobilized copper ion affinity membranes. J.Chromatogr. A, 2003,996 (1): 53-70
[32] Q.-H. Shi. Chitosan-coated silica beads as immobilized metal affinity support for protein adsorption.Biochem.Engin.J.,2003,16 (3):317-322
[33] Fengna Xi, Macroporous chitosan layer coated on non-porous silica gel as a support for metal chelate affinity chromatographic adsorbent. J.Chromatogr. A, 2004,1057 (1): 41-47
[34] Sibel Emir, Ridvan Say.A New Metal Chelate Affinity Adsorbent for Cytochrome C.Biotechnol. Prog. 2004, 20(1):223-228
[35] Sinan Akgol. Novel metal-chelate affinity sorbents for reversible use in catalase adsorption. J.Molecu.Cata.B: Enzymatic ,2004,28 (1): 7-14
[36] Adil Denizli, Novel metal-chelate affinity adsorbent for purification of immunoglobulin-G from human plasma, J.Chromatogr.B, 795 (2003) 93-103
[37] M Zaveckas, B Baskeviciute,V Luksa. Comparative studies of recombinant human granulocyte-colony stimulating factor, its Ser-17 and (His)6-tagged forms interaction with metal ions by means of immobilized metal ion affinity partitioning: Effect of chelated nickel and mercuric ions on extraction and refolding of proteins from inclusion bodies. J.Chromatogr. A ,2000,904(2): 145—169.
[38] M Zaveckas, M T Heam. Adsorption and selectivity characteristics of several human serum proteins with immobilised hard Lewis metal ion-chelate adsorbents. J.Chromatogr.A, 2000, 890(1):95-116.
[39] Gulay Bayramoglu. Effect of spacer-arm and Cu(II) ions on performance of 1-histidine immobilized on poly(GMA/MMA) beads as an affinity ligand for separation and purification of IgG.Sep. Pur.Tech,2006, 50(2) :229-239
[40]R M CHicz,F E Renier.Immobilized-metal affinity and hydroxyapatite chromatography of genetically engineered subtilisin. Anal.Chem., 1989,61 (15): 1742- 1749.
[41]Serpa, Gisele; Augusto.Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody. J.Chromatogr.B,2005, 816(2): 259-268
[42] Zhi-Ya Ma,Xian-Qiao Liu. Synthesis of magnetic silica nanospheres with metal ligands and application in affinity separation of proteins.Colloids Surf. A,2006, 275(1-3):87-91
[43] V. Gupta, A.N.S.Eshwari. Optimization of immobilized metal ion affinity chromatography for single-step purification of recombinant ovine growth hormone J.Chromatogr.A, 2003, 998(1):93-101
[44] Quanzhou Luo,Hanfa Zou. Chromatographic separation of proteins on metal immobilized iminodiacetic acid-bound molded monolithic rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate). J.Chromatogra. A,2001,926(2):255-264
[45] Denizli A., Piskin E. Dye-ligand affinity systems.J. Biochem. Biophys. Methods,2001, 49:391-416
[46] Hey Y, Dean P.D.G.. Dyes.A colorful addition to protein purification.Chem.Indus., 1981,20:726-732
[47] Hua—Li Nie, Li—Min Zhu. Adsorption of papain with Cibacron Blue F3GA carrying chitosan-coated nylon affinity membranes. Int.J. Biol. Macromol, 2007,40 (3) :261-267
[48]Lowe.C.R,Small D.A.P., Atkinson T.Some preparative and analytical applications of triazine dyes. Inter.J.Biochem.,1981,13(1):33-40
[49] Tsamadis G., papageorgakopoulou N., Clonis Y.D. Downstream processing of diagnostic enzymes :optimized protocls for the simultaneous separation and purification of lactate dehydrogenase and pyruvate kinase from rabbit muscle.Bioproc Engin,1992,7(5):213-218
[50] Katsos N.E., Labrou N.E., Clonis Y.D. Interaction of 1-glutamate oxidase with triazine dyes: selection of ligands for affinity chromatography. J. Chromatogr. B,2004,807(2) :277-285
[51] Ruckenstein E., Zeng X. F. Albumin separation with Cibacron Blue carrying macroporous chitosan and chitin affinity membranes. J. Membr. Sci., 1998, 142(1): 13-26
[52] Labrou N. E., Clonis Y. D. Biomimetic dye affinity chromatography for the purification of bovine heart lactate dehydrogenase. Journal of chromatographyA, 1995, 718(1): 35-44
[53] Handan Yacuz, Sinan Akgol. Affinity separation of immunoglobulin G subclasses on dye attached poly(hydroxypropyl methacrylate) beads. Int. J. Biol. Macromol, 2006, 39 (4-5: 303-309
[54] Ohlson. S. Hamsson L. Larsom P O. High performance liquid affinity chromatography(HPLAC) and its application to separation of enzymes and antigens. FEBS Lett., 1978, 93(1): 5-9
[55] Luis A. Jurado. In flow activation of diol-silica with cyanogen bromide and triethylamine for preparing high-performance affinity chromatographic columns. J. Chromatogr. A, 2003, 984 (1): 9-17
[56] David S. Hage. High-performance affinity chromatography and immobilized serum albumin as probes for drug- and hormone-protein binding. J. Chromatogr. B, 2000, 739 (1): 39-54
[57] Rui Zhaoa. Screening of inhibitors for influenza A virus using high-performance affinity chromatography and combinatorial peptide libraries. J. Chromatogr. A, 2005, 1064 (1): 59-66
[58] 郭立安，抗rhIL-2单克隆抗体高效亲和色谱填料的制备及其应用，第四军医大学学报，2000，21(10)：1195-1196
[59] 赵莹歆，一种分离抗凝血酶的高效亲和色谱填料的制备及表征，高等学校化学学报，2002，23(2)：203-206
[60] 余晓，以寡聚组氨酸为配基的高效亲和色谱研究，高等学校化学学报，2000，21(4)：532-534
[61] 吴晓军，磺胺甲基异溅唑作配基的高效亲和色谱分离胰凝乳蛋白酶和胰蛋白酶，分析化学研究报告，1996，24(11)：1245-1247
[62] Zeng Q. B., Xu J. R., Fu R. W., Journal of Functional Polymers, 2000, 13(3): 333-348
[63] Nawrocki J., Rigney M.P., Mcormick A. Chemistry of zirconia and its use in chromatography. J Chromatogr., 1993, 657(2): 229-82
[64] Afeyan N. B., Fulton S. P., Regnier F. E. Perfusion chromatography packing materials for proteins and peptides. J Chromatogr, 1991, 544(1-2): 267-279
[65] Dominic C. Nash., Graham E, McCreath, Howard A. Modification ofpolustyrenic matrixed for the purification of proteins. Effect of the adsorption of poly(vinyl alcohol) on the characteristics of poly(styrene-divinybenzene) beads for use in affinity chromatography. J. Chromatogr. A, 1997, 758(1): 53-64
[66] 王佳兴，生物化学中应用的离子交换介质简介，离子交换与吸附，1992，8(4)：366-373
[67] Zhi-Ya Ma, Xian-Qiao Liu. Synthesis of magnetic silica nanospheres with metal ligands and application in affinity separation of proteins. Colloids Surf. A, 2006, 275(1-3): 87-91
[68] Renwei Wang, Ying Zhang. Modification of poly(glycidyl methacrylate-divinylbenzene) porous microspheres with polyethylene glycol and their adsorption property of protein. Colloids Surf, B, 2006, 51 (1): 93-99
[69] Kondo A., et al. Development and application ofthermo-sensitive immunomicrospheres for antibody purification. Biotech. Bioengin., 1994, 44(1): 1-6
[70] Zhu Y. H., Wang Q. B., et al. Preparation of Surface Functional Magnetic Microspheres and Their Application in Nucleic Acid Separation and Enzyme Immobilization. Zhongguo Yixue Kexueyuan Xuebao, 2002, 24(2): 118-123
[71] Alena S., Bohusiav R., et al. Immunomagnetic separation and detection of Salmonella cells using newly designed carriers. J. Chromatogr. A, 2003, 1009 (1-2): 215-221
[72] Jana K., Alena S., et al. Magnetic hydrophilic methacrylate-based polymer microspheres for genomic DNA isolation. J. Chromatogr. A, 2005, 1064(2): 247-253
[73] Yakup A. M., Emine Y., Guelay B. Polyethylenimine-grafted and HSA-immobilized poly(GMA-MMA) affinity adsorbents for bilirubin removal. Polym. Inter., 2005, 54(1): 153-160
[1] 于世林，高效液相色谱方法及应用，化学工业出版社，2000
[2] Ogino K., Sato H.. Synthesis ofmonodisperse macroreticular styrene-divinylbenzene gel particles by a single-step swelling and polymerization method. J. Chromatogr. A, 1995, 699(1): 59-66
[3] Ogino K., Sato H., et al. Preparation and characterization of monodisperse oligo(ethylene glycol) dimethacrylate polymer beads for aqueous high-performance liquid chromatography. J. Chromatogr. A, 1995, 699(1): 67-72
[4] 吴飞燕．染料配基亲和色谱填料制备：[硕士学位论文]．杭州：浙江大学，2005
[5] 吴承佣，周彩华，高分子化学实验，1989年
[1] 邸泽梅，陈国亮，金属螯合亲和色谱介质的合成及其色谱特性研究，色谱，1998，16(4)：297-300
[2] Feiyan Wu, Yan Zhu. Preparation of dye-ligand affinity chromatographic packings based on monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their chromatographic properties. J. Chromatogra. A, 2006, 1134 (1-2): 45-50
[1] He LZ, Gan YR. Adsorption-desorption of BSA to highly substituted dye-ligand adsorbent:quantitative study of the effect of ionic strength. Bioproc Eng, 1997,17(5):301 -305
[2] Subramanian S. Spectral changes induced in Cibacron blue F3GA by salts,organic solvents,and polypeptides:implications for blue dye interaction with proteins. Arch Biochem Biophys, 1982,216:116-125
[3] H Y Reina, A R Manriquez. Selectivity of IMAC Column in Trypsin nhibitor Purification. Biotechno. Prog., 2001, 17 (4): 729-733
[4] F B Anspach.Silica-based metal chelate affinity sorbents.Adsorption and elution behavior of proteins on iminodiacetic acid affinity sorbents prepared via different immobilization techniques. J.Chromatogr., 1994, 676 (2); 249-266
[2] 唐欢欢，王林，亲和色谱在医药生物中的应用，医药化工，2005，(5)：24-29
[3] 韩金玉，那平．亲和色谱纯化蛋白质新进展，色谱，1996，14(6)；447-450
[4] Cuatrecasas P., Wilchek M., Single-step purification of avidine from egg white by affinity chromatography on biocytin-Sepharose columns. Biochem. Biophys. Res. Commun, 1968, 33(2): 235-244
[5] Denizli A., Piskin E. Dye-ligand affinity systems. J. Biochem. Biophys. J. Biochem. Biophys. Methods, 2001, 49: 391-416
[6] 任军，贾凌云，亲和色谱仿生配基的筛选和设计，分析化学，2005，(9)：1345-1349
[7] 周绪斌，宣华，李荣秀，组特异性亲和配基在分离纯化中的应用，中国生化药杂志 2000，21(6)：308-310
[8] Ozkara S, Yacuz H, Denizli A. Purification of immunoglobulin G from human plasma by metal-chelate affinity chromatography, J. Appl. Polym. Sci., 2003, 89 (6): 1567-1572
[9] Lowe C R, Pearson J C. Affinity chromatography on immobilized dyes. Methods Enzymol., 1984, 104: 97-113
[10] Clonis Y D, Labrou N E, Kotsira V, Mazitsos K. Biomimetic dyes as affinity chromatography tools in enzyme purification J. Chromatogr. A, 2000, 891 (1); 33-44
[11] Starkenstein E., Biochem. Z, 1910, 24: 210-218
[12] Cuatrecasas P., Wilchek M., Anfinsen C. B. Selective enzyme purification by affinity chromatography. Proc. Nat. Acad. Sci. U S., 1968, 61(2): 636-643
[13] Livshits M A, Mirzabekov A D. Theoretical analysis of the kinetics of DNA hybridization with gel-immobilized oligonucleotides. Biophys J., 1996, 71(5): 2795-2801
[14] Jamn J.The kinetics of protein-protein recognition. Proteins: Struct Funct Genet, 1997, 28(2): 153-161
[15] Meir Wilchek, Talia Miron, Thirty years of affinity chromatography. Reactive &Funct. polym, 1999, 41: 263-268
[16] Bonnerjea J., Dh s., Hoare M. Protein purification: the right step at the right time. Biotech, 1986, 4: 954-958
[17] Groman E. V., Wilchek M. Recent developments in affinity chromatography supports. Trends Biotech, 1987, 5 (8): 220-224
[18] Tjonnfjord G E, Steen R,Veiby O.P. Lineage commitment of CD34+ human hematopoietic progenitor cells. Exp Hematol, 1996, 24(5): 875—882
[19] Liang X., Lubman D. M., Ross D. T. On-Probe Immunoaffinity Extraction by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Anal Chem, 1998, 70(3): 498-503
[20] Porath J, Carlsson J, Olsson I, Belfrage G. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature, 1975, 258(5536): 598
[21] 李蓉，陈国亮，赵文明．蛋白质在合成阳离子交换剂上的色谱特性研究色谱，2004，22(2)：134-137．
[22] J W Wong, R L Albright, N H L Wang.Immobilized metal ion affinity chromatography(IMAC) chemistry and bioseparation application. Separation and Purification Methods, 1991, 20 (1): 49-106
[23] H Y Reina, A R Manriquez, B R Wong, Selectivity of IMAC Columns in Trypsin Inhibitor Purification. Biotechnol. Prog., 2001, 17(4): 729-733
[24] Pearson, R. G. In: Rearson, R. G., editor. Hard and soft acids and bases. Hutchington &Ross, 1973. p. 53-9, 67-85.
[25] Grigoriy S. Chaga. Twenty-five years of immobilized metal ion affinity chromatography:past,present, and future.J.Biochem.Biophys.Methods 49(2001)313-334
[26] Z. Y. Ma, Y. P. Guan, Synthesis of Magnetic Chelator for High-Capacity Immobilized Metal Affinity Adsorption of Protein by Cerium Initiated Graft Polymerization.Langmuir,2005, 21(15), 6987-6994
[27] Aurelio Hidalgo, Chlorpromazine N-Demethylation by Hydroperoxidawd Activity of Covalent Immobilized Lipoxygenase.Biotechnol. Prog. 2004, 20(5), 1578—1582
[28] Luciana Cristina Lins de Aquino, Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin . J.Chromatogr.B, 2006,834(1): 68-76
[29] MARTIN LUND.Purification and Identification of Water-Soluble Phosphopeptides from Cheese Using Fe(III) Affinity Chromatography and Mass Spectrometry. J. Agric. Food Chem. 2004, 52, 6616-6622
[30] M. Yakup Aryca. Dye-ligand and metal chelate poly(2-hydroxyethylmethacrylate) membranes for affinity separation of proteins. J.Chromatogr. A, 1998,799(1): 83-91
[31] Chun-Yi Wu. Analysis of protein adsorption on regenerated cellulose-based immobilized copper ion affinity membranes. J.Chromatogr. A, 2003,996 (1): 53-70
[32] Q.-H. Shi. Chitosan-coated silica beads as immobilized metal affinity support for protein adsorption.Biochem.Engin.J.,2003,16 (3):317-322
[33] Fengna Xi, Macroporous chitosan layer coated on non-porous silica gel as a support for metal chelate affinity chromatographic adsorbent. J.Chromatogr. A, 2004,1057 (1): 41-47
[34] Sibel Emir, Ridvan Say.A New Metal Chelate Affinity Adsorbent for Cytochrome C.Biotechnol. Prog. 2004, 20(1):223-228
[35] Sinan Akgol. Novel metal-chelate affinity sorbents for reversible use in catalase adsorption. J.Molecu.Cata.B: Enzymatic ,2004,28 (1): 7-14
[36] Adil Denizli, Novel metal-chelate affinity adsorbent for purification of immunoglobulin-G from human plasma, J.Chromatogr.B, 795 (2003) 93-103
[37] M Zaveckas, B Baskeviciute,V Luksa. Comparative studies of recombinant human granulocyte-colony stimulating factor, its Ser-17 and (His)6-tagged forms interaction with metal ions by means of immobilized metal ion affinity partitioning: Effect of chelated nickel and mercuric ions on extraction and refolding of proteins from inclusion bodies. J.Chromatogr. A ,2000,904(2): 145—169.
[38] M Zaveckas, M T Heam. Adsorption and selectivity characteristics of several human serum proteins with immobilised hard Lewis metal ion-chelate adsorbents. J.Chromatogr.A, 2000, 890(1):95-116.
[39] Gulay Bayramoglu. Effect of spacer-arm and Cu(II) ions on performance of 1-histidine immobilized on poly(GMA/MMA) beads as an affinity ligand for separation and purification of IgG.Sep. Pur.Tech,2006, 50(2) :229-239
[40]R M CHicz,F E Renier.Immobilized-metal affinity and hydroxyapatite chromatography of genetically engineered subtilisin. Anal.Chem., 1989,61 (15): 1742- 1749.
[41]Serpa, Gisele; Augusto.Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody. J.Chromatogr.B,2005, 816(2): 259-268
[42] Zhi-Ya Ma,Xian-Qiao Liu. Synthesis of magnetic silica nanospheres with metal ligands and application in affinity separation of proteins.Colloids Surf. A,2006, 275(1-3):87-91
[43] V. Gupta, A.N.S.Eshwari. Optimization of immobilized metal ion affinity chromatography for single-step purification of recombinant ovine growth hormone J.Chromatogr.A, 2003, 998(1):93-101
[44] Quanzhou Luo,Hanfa Zou. Chromatographic separation of proteins on metal immobilized iminodiacetic acid-bound molded monolithic rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate). J.Chromatogra. A,2001,926(2):255-264
[45] Denizli A., Piskin E. Dye-ligand affinity systems.J. Biochem. Biophys. Methods,2001, 49:391-416
[46] Hey Y, Dean P.D.G.. Dyes.A colorful addition to protein purification.Chem.Indus., 1981,20:726-732
[47] Hua—Li Nie, Li—Min Zhu. Adsorption of papain with Cibacron Blue F3GA carrying chitosan-coated nylon affinity membranes. Int.J. Biol. Macromol, 2007,40 (3) :261-267
[48]Lowe.C.R,Small D.A.P., Atkinson T.Some preparative and analytical applications of triazine dyes. Inter.J.Biochem.,1981,13(1):33-40
[49] Tsamadis G., papageorgakopoulou N., Clonis Y.D. Downstream processing of diagnostic enzymes :optimized protocls for the simultaneous separation and purification of lactate dehydrogenase and pyruvate kinase from rabbit muscle.Bioproc Engin,1992,7(5):213-218
[50] Katsos N.E., Labrou N.E., Clonis Y.D. Interaction of 1-glutamate oxidase with triazine dyes: selection of ligands for affinity chromatography. J. Chromatogr. B,2004,807(2) :277-285
[51] Ruckenstein E., Zeng X. F. Albumin separation with Cibacron Blue carrying macroporous chitosan and chitin affinity membranes. J. Membr. Sci., 1998, 142(1): 13-26
[52] Labrou N. E., Clonis Y. D. Biomimetic dye affinity chromatography for the purification of bovine heart lactate dehydrogenase. Journal of chromatographyA, 1995, 718(1): 35-44
[53] Handan Yacuz, Sinan Akgol. Affinity separation of immunoglobulin G subclasses on dye attached poly(hydroxypropyl methacrylate) beads. Int. J. Biol. Macromol, 2006, 39 (4-5: 303-309
[54] Ohlson. S. Hamsson L. Larsom P O. High performance liquid affinity chromatography(HPLAC) and its application to separation of enzymes and antigens. FEBS Lett., 1978, 93(1): 5-9
[55] Luis A. Jurado. In flow activation of diol-silica with cyanogen bromide and triethylamine for preparing high-performance affinity chromatographic columns. J. Chromatogr. A, 2003, 984 (1): 9-17
[56] David S. Hage. High-performance affinity chromatography and immobilized serum albumin as probes for drug- and hormone-protein binding. J. Chromatogr. B, 2000, 739 (1): 39-54
[57] Rui Zhaoa. Screening of inhibitors for influenza A virus using high-performance affinity chromatography and combinatorial peptide libraries. J. Chromatogr. A, 2005, 1064 (1): 59-66
[58] 郭立安，抗rhIL-2单克隆抗体高效亲和色谱填料的制备及其应用，第四军医大学学报，2000，21(10)：1195-1196
[59] 赵莹歆，一种分离抗凝血酶的高效亲和色谱填料的制备及表征，高等学校化学学报，2002，23(2)：203-206
[60] 余晓，以寡聚组氨酸为配基的高效亲和色谱研究，高等学校化学学报，2000，21(4)：532-534
[61] 吴晓军，磺胺甲基异溅唑作配基的高效亲和色谱分离胰凝乳蛋白酶和胰蛋白酶，分析化学研究报告，1996，24(11)：1245-1247
[62] Zeng Q. B., Xu J. R., Fu R. W., Journal of Functional Polymers, 2000, 13(3): 333-348
[63] Nawrocki J., Rigney M.P., Mcormick A. Chemistry of zirconia and its use in chromatography. J Chromatogr., 1993, 657(2): 229-82
[64] Afeyan N. B., Fulton S. P., Regnier F. E. Perfusion chromatography packing materials for proteins and peptides. J Chromatogr, 1991, 544(1-2): 267-279
[65] Dominic C. Nash., Graham E, McCreath, Howard A. Modification ofpolustyrenic matrixed for the purification of proteins. Effect of the adsorption of poly(vinyl alcohol) on the characteristics of poly(styrene-divinybenzene) beads for use in affinity chromatography. J. Chromatogr. A, 1997, 758(1): 53-64
[66] 王佳兴，生物化学中应用的离子交换介质简介，离子交换与吸附，1992，8(4)：366-373
[67] Zhi-Ya Ma, Xian-Qiao Liu. Synthesis of magnetic silica nanospheres with metal ligands and application in affinity separation of proteins. Colloids Surf. A, 2006, 275(1-3): 87-91
[68] Renwei Wang, Ying Zhang. Modification of poly(glycidyl methacrylate-divinylbenzene) porous microspheres with polyethylene glycol and their adsorption property of protein. Colloids Surf, B, 2006, 51 (1): 93-99
[69] Kondo A., et al. Development and application ofthermo-sensitive immunomicrospheres for antibody purification. Biotech. Bioengin., 1994, 44(1): 1-6
[70] Zhu Y. H., Wang Q. B., et al. Preparation of Surface Functional Magnetic Microspheres and Their Application in Nucleic Acid Separation and Enzyme Immobilization. Zhongguo Yixue Kexueyuan Xuebao, 2002, 24(2): 118-123
[71] Alena S., Bohusiav R., et al. Immunomagnetic separation and detection of Salmonella cells using newly designed carriers. J. Chromatogr. A, 2003, 1009 (1-2): 215-221
[72] Jana K., Alena S., et al. Magnetic hydrophilic methacrylate-based polymer microspheres for genomic DNA isolation. J. Chromatogr. A, 2005, 1064(2): 247-253
[73] Yakup A. M., Emine Y., Guelay B. Polyethylenimine-grafted and HSA-immobilized poly(GMA-MMA) affinity adsorbents for bilirubin removal. Polym. Inter., 2005, 54(1): 153-160
[1] 于世林，高效液相色谱方法及应用，化学工业出版社，2000
[2] Ogino K., Sato H.. Synthesis ofmonodisperse macroreticular styrene-divinylbenzene gel particles by a single-step swelling and polymerization method. J. Chromatogr. A, 1995, 699(1): 59-66
[3] Ogino K., Sato H., et al. Preparation and characterization of monodisperse oligo(ethylene glycol) dimethacrylate polymer beads for aqueous high-performance liquid chromatography. J. Chromatogr. A, 1995, 699(1): 67-72
[4] 吴飞燕．染料配基亲和色谱填料制备：[硕士学位论文]．杭州：浙江大学，2005
[5] 吴承佣，周彩华，高分子化学实验，1989年
[1] 邸泽梅，陈国亮，金属螯合亲和色谱介质的合成及其色谱特性研究，色谱，1998，16(4)：297-300
[2] Feiyan Wu, Yan Zhu. Preparation of dye-ligand affinity chromatographic packings based on monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads and their chromatographic properties. J. Chromatogra. A, 2006, 1134 (1-2): 45-50
[1] He LZ, Gan YR. Adsorption-desorption of BSA to highly substituted dye-ligand adsorbent:quantitative study of the effect of ionic strength. Bioproc Eng, 1997,17(5):301 -305
[2] Subramanian S. Spectral changes induced in Cibacron blue F3GA by salts,organic solvents,and polypeptides:implications for blue dye interaction with proteins. Arch Biochem Biophys, 1982,216:116-125
[3] H Y Reina, A R Manriquez. Selectivity of IMAC Column in Trypsin nhibitor Purification. Biotechno. Prog., 2001, 17 (4): 729-733
[4] F B Anspach.Silica-based metal chelate affinity sorbents.Adsorption and elution behavior of proteins on iminodiacetic acid affinity sorbents prepared via different immobilization techniques. J.Chromatogr., 1994, 676 (2); 249-266