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通过含有功能化聚合物刷的亲和膜对His标签蛋白进行高容量纯化。

High-capacity purification of His-tagged proteins by affinity membranes containing functionalized polymer brushes.

作者信息

Jain Parul, Sun Lei, Dai Jinhua, Baker Gregory L, Bruening Merlin L

机构信息

Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.

出版信息

Biomacromolecules. 2007 Oct;8(10):3102-7. doi: 10.1021/bm700515m. Epub 2007 Sep 19.

Abstract

Porous membrane absorbers are attractive for increasing the rate of protein purification, but their binding capacity is low relative to porous beads. Modification of membranes with functionalized polymer brushes, however, can greatly enhance capacity. This work demonstrates that membrane modification with poly(2-hydroxyethyl methacrylate) (PHEMA) brushes derivatized with nitrilotriacetate-Ni2+ (NTA-Ni2+) complexes allows purification of polyhistidine-tagged ubiquitin (HisU) in less than 30 min with a binding capacity of 120 mg of HisU/cm3 of porous alumina membrane. Adsorption isotherms show that saturation of the brushes occurs at HisU concentrations as low as 0.04 mg/mL and that these brushes can bind up to 23 monolayers of HisU. Gel electrophoresis reveals that the purity of eluted HisU is more than 99%, even when the initial feed solution contains 10% bovine serum or a 20-fold excess of BSA. Thus, reusable porous membranes modified by PHEMA-NTA-Ni2+ brushes are attractive candidates for rapid purification of polyhistidine-tagged proteins.

摘要

多孔膜吸附剂对于提高蛋白质纯化速率具有吸引力,但其结合能力相对于多孔珠而言较低。然而,用功能化聚合物刷对膜进行修饰可大大提高其容量。这项工作表明,用衍生有次氮基三乙酸-Ni2+(NTA-Ni2+)配合物的聚甲基丙烯酸2-羟乙酯(PHEMA)刷修饰膜,能够在不到30分钟的时间内纯化多聚组氨酸标记的泛素(HisU),多孔氧化铝膜的结合容量为120 mg HisU/cm3。吸附等温线表明,在低至0.04 mg/mL的HisU浓度下刷就会饱和,并且这些刷最多可结合23个HisU单分子层。凝胶电泳显示,即使初始进料溶液含有10%的牛血清或20倍过量的牛血清白蛋白(BSA),洗脱的HisU纯度仍超过99%。因此,用PHEMA-NTA-Ni2+刷修饰的可重复使用的多孔膜是快速纯化多聚组氨酸标记蛋白质的有吸引力的候选材料。

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