Thermosensitive copolymers of N-vinylimidazole as displacers of proteins in immobilised metal affinity chromatography
Thermosensitive copolymers of N-vinylimidazole as displacers of proteins in immobilised metal affinity chromatography
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DOI：
10.1016/s0021-9673(00)00959-6
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发表时间：
2001-01-12
影响因子：
4.1
通讯作者：
Mattiasson, B
中科院分区：
文献类型：
--
作者：
Ivanov, AE;Galaev, IY;Mattiasson, B
合成了N-乙烯基己内酰胺（VCL）和N-乙烯基咪唑（VI）的共聚物，研究了其作为蛋白质固定化金属亲和色谱（IMAC）的温敏、可重复使用的置换剂。以9：1的单体摩尔比通过自由基聚合制备的重均分子量为11 700 g/mol的共聚物通过IMAC和热沉淀分离成几个级分。在Cu ~（2+）-IDA-Sepharose上，平均VI含量为8.5%的组分作为卵白色蛋白、溶菌酶等蛋白质的IMAC的重复使用置换剂效果最好。置换剂表现出尖锐的穿透曲线和16-20 mg/ml凝胶的结合容量，取决于流速。在置换层析过程中，鸡蛋白色蛋白的回收率> 95%。在48 ℃下通过热沉淀可从蛋白质组分中定量除去置换剂。在3%（v/v）乙腈存在下，溶菌酶与置换剂的共沉淀最小，而上清液中的溶菌酶酶活性完全保留。向移动的相中加入游离咪唑可提高蛋白质解吸速率，并可在色谱过程中更好地分离鸡蛋白色蛋白和置换剂。蛋白白色提取物的置换谱由三个区域组成，通过SDS-PAGE表征各个蛋白质的不同分布。用0.15 M氯化钠（pH 8.0）中的0.02 M EDTA轻松进行色谱柱再生，然后用蒸馏水洗涤并用Cu 2+重新加载。置换剂也可以通过在48 ℃下热沉淀和随后用稀盐酸（pH 2.5）透析来再生。(C)2001 Elsevier Science B. V.保留所有权利。
Synthetic copolymers of N-vinylcaprolactam (VCL) and N-vinylimidazole (VI) were studied as thermosensitive, reusable displacers for immobilised metal affinity chromatography (IMAC) of proteins. The copolymer with weight-average molecular mass of 11 700 g/mol prepared by free radical polymerisation at a 9:1 monomer molar ratio was separated into several fractions by IMAC and thermal precipitation. The fraction with an average VI content of 8.5% was most efficient as a reusable displacer For IMAC of ovalbumin, lysozyme and other proteins of egg white on Cu2+-IDA-Sepharose. The displacer exhibited a sharp breakthrough curve and binding capacity of 16-20 mg/ml gel, depending on the flow-rate. The recovery of egg white proteins in the course of displacement chromatography was >95%. The displacer could be removed quantitatively from the protein fractions by thermal precipitation at 48 degreesC. Co-precipitation of lysozyme with the displacer was minimal in the presence of 3% (v/v) acetonitrile, while the lysozyme enzymatic activity in the supernatant was completely retained. Addition of free imidazole to the mobile phase increased the rate of protein desorption and allowed better separation of egg white proteins and the displacer in the course of chromatography. The displacement profile of the egg white extract consisted of three zones with different distributions of individual proteins characterised by SDS-PAGE. Regeneration of the column was easily performed with 0.02 M EDTA in 0.15 M sodium chloride, pH 8.0, followed by washing with distilled water and reloading with Cu2+. The displacer could also be regenerated by thermal precipitation at 48 degreesC and subsequent dialysis against dilute hydrochloric acid (pH 2.5). (C) 2001 Elsevier Science B.V. All rights reserved.