基于磁性微球的固定化金属亲和载体及其在蛋白质/多肽纯化中的应用

采用模板法制备的单分散磁性硅胶微球,经过表面修饰偶联上亚氨基二乙酸(IDA),与过渡金属离子Cu2 螯合,制成一种新型的磁性固定化金属亲和纯化载体。用牛血清白蛋白(BSA)作为模型进行磁性固定化金属亲和吸附蛋白的研究,结果表明,BSA在磁性亲和载体上的吸附可用Langmuir吸附方程描述,对BSA的饱和吸附量为90mg/g。将磁性亲和载体用于带有组氨酸标签的镇痛抗肿瘤多肽(analgesic-antitumorpeptide,AGAP)纯化,在未经过滤的细胞裂解液中可以将AGAP一步纯化,非特异性吸附低,操作简便,完全适用于含有组氨酸标记的重组多肽或蛋白的分离纯化。

Magnetic Silica Microspheres as a Novel Support for Immobilized Metal Affinity Purification of Proteins/Peptides

Magnetic silica microbeads with high magnetization,uniform particle size distribution were modified by silane coupling agents to yield a novel support for immobilized metal affinity purification of proteins.The synthetic procedure consists of three steps:(i)the preparation of magnetic silica microbeads by polymer templating method;(ii)coupling of iminodiacetic acid(IDA)with a bifunctional silane agent 3-glycidoxypropyltrimethoxysilane;(iii)charging the magnetic support with Cu2 .With bovine serum albumin(BSA)as a model compound,the adsorption behavior of protein on the magnetic matrix was studied.Preliminary results show that the adsorption of BSA can be described by Langmuir equation with saturation capacity being 90 mg/g.These magnetic microbeads were further applied to purify a recombinant peptide Analgesic-antitumor peptide(AGAP),directly from cell lysis without sample pretreatment.Being simple and specific,the magnetic silica microbeads based protocol is especially suited for purification of histidine-tagged recombinant peptides or proteins.