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北京石油化工学院 新材料与化工学院,北京 102617
Received:19 November 2024,
Revised:15 January 2025,
Accepted:20 January 2025,
Published Online:13 June 2025,
Published:15 July 2025
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侯恒扬,李子炀,乔娟,马磊,靳海波,张荣月.接枝法制备大孔金属固定化亲和层析介质及其蛋白分离性能研究[J].分析测试学报,2025,44(07):1-10.
HOU Heng-yang,LI Zi-yang,QIAO Juan,MA Lei,JIN Hai-bo,ZHANG Rong-yue.Preparation of Macroporous Metal Immobilized Affinity Chromatography Media by Grafting Method and Evaluation of Its Protein Separation Performance[J].Journal of Instrumental Analysis,2025,44(07):1-10.
侯恒扬,李子炀,乔娟,马磊,靳海波,张荣月.接枝法制备大孔金属固定化亲和层析介质及其蛋白分离性能研究[J].分析测试学报,2025,44(07):1-10. DOI: 10.12452/j.fxcsxb.241119540.
HOU Heng-yang,LI Zi-yang,QIAO Juan,MA Lei,JIN Hai-bo,ZHANG Rong-yue.Preparation of Macroporous Metal Immobilized Affinity Chromatography Media by Grafting Method and Evaluation of Its Protein Separation Performance[J].Journal of Instrumental Analysis,2025,44(07):1-10. DOI: 10.12452/j.fxcsxb.241119540.
该文以大孔聚丙烯酸酯微球为基质,甲基丙烯酸缩水甘油酯-亚氨基二乙酸(GMA-IDA)功能单体为配基,通过氧化还原反应,制备了金属亲和层析介质。首先,基于GMA的环氧基团与IDA的亚氨基反应制备GMA-IDA功能单体,随后通过氧化还原反应,使功能单体中的双键进行接枝聚合制备大孔聚合物微球。最后在0.1 mol/L镍溶液下螯合Ni
2+
,制备FastSep-GMA-IDA-Ni亲和层析介质。考察了接枝聚合反应中各因素对蛋白吸附容量的影响,包括功能单体浓度、引发剂浓度、反应体系pH值、反应温度、反应时间等,确定了Fastsep-GMA-IDA-Ni亲和层析介质的最佳制备条件。以牛血红蛋白为模型蛋白,所得FastSep-GMA-IDA-Ni亲和介质的静态结合容量最高可达47.97 mg/mL,比商品Ni亲和层析介质高约25.75%。通过偶联法制备的FastSep-IDA-Ni亲和层析介质,其静态结合容量为1.652 7 mg/mL,远低于接枝法制备的FastSep-GMA-IDA-Ni亲和层析介质。且FastSep-GMA-IDA-Ni相比商品介质显示出更好的蛋白传质效率。考察发现,随着镍含量的增加,同种条件下制备的Fastsep-GMA-IDA-Ni亲和层析介质的蛋白静态结合容量随之增加直至达到饱和。结果显示,随着离子交换容量的增加,螯合的镍含量也随之增加,最高镍含量为156.6 μmol/mL。将制备的FastSep-GMA-IDA-Ni对CHO细胞表达的血凝素(HA-His)蛋白进行分离纯化,得到了良好的分离效果。
Immobilized metal affinity chromatography medium is widely used in the downstream processing of proteins due to their high purity and low preparation cost. The metal affinity chromatography medium which was based on the macroporous polyacrylate microspheres,was prepared by redox reaction using glycidyl methacrylate-iminodiacetic acid(GMA-IDA) functional monomer as ligand. Firstly,the GMA-IDA functional monomer is prepared by the reaction of the epoxy group of GMA with the imino of IDA. Seco
ndly,the double bonds in the functional monomer are grafted to prepare macroporous polymer microspheres by redox reaction. Finally,the FastSep-GMA-IDA-Ni affinity chromatography medium was prepared by chelating Ni
2+
in 0.1 mol/L nickel solution. The influence of various factors on protein binding capacity of graft polymerization was investigated,including functional monomer concentration,initiator concentration,reaction system pH,reaction temperature,reaction time,etc. The optimum preparation conditions of Fastsep-GMA-IDA-Ni affinity chromatography were determined. Using bovine hemoglobin as the model protein,the static binding capacity of FastSep-GMA-IDA-Ni affinity medium was 47.97 mg/mL,which was about 25.75% higher than that of commercial Ni affinity chromatography medium. FastSep-IDA-Ni affinity chromatography medium was prepared by coupling method with a static binding capacity of 1.652 7 mg/mL,which was much lower than that of FastSep-GMA-IDA-Ni affinity chromatography medium prepared by grafting method. Moreover,the mass transfer rate
k
2
value of FastSep-GMA-IDA-Ni is 0.001 18 mL/mg/min,and the mass transfer rate
k
2
value of commercial medium is 0.000 74 mL/mg/min,indicating that FastSep-GMA-IDA-Ni has better protein mass transfer efficiency than commercial medium. The relationship between nickel content and protein static binding capacity in Fastsep-GMA-IDA-Ni affinity chromatography was observed. The nickel content of chelate increased with the increase of ion-exchange capacity. The highest nickel content was 156.6 μmol/mL. The prepared FastSep-GMA-IDA-Ni was used to separate and purify the hemagglutinin(HA-His) protein expressed by CHO cells. The results showed that the medium has good separation performance.
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