Arvidsson P, Ivanov A E, Mattiasson B
Department of Biotechnology, Center for Chemistry and Chemical Engineering Lund University, Sweden.
J Chromatogr B Biomed Sci Appl. 2001 Apr 5;753(2):279-85. doi: 10.1016/s0378-4347(00)00558-2.
Successful immobilized metal affinity chromatography (IMAC) of proteins on Cu2+-iminodiacetic acid Sepharose has been carried out in a displacement mode using a synthetic copolymer of vinyl imidazole and vinyl caprolactam [poly(VI-VCL)] as a displacer. Vinyl caprolactam renders the co-polymer with the thermosensitivity, e.g., property of the co-polymer to precipitate nearly quantitatively from aqueous solution on increase of the temperature to 48 degrees C. A thermostable lactate dehydrogenase from the thermophilic bacterium Bacillus stearothermophilus modified with a (His)6-tag [(His)6-LDH] has been purified using an IMAC column. For the first time it was clearly demonstrated that a polymeric displacer [poly(VI-VCL)] was more efficient compared to a monomeric displacer (imidazole) of the same chemical nature, probably due to the multipoint interaction of imidazole groups within the same macromolecule with one Cu2+ ion. Complete elution of bound (His)6-LDH has been achieved at 3.7 mM concentration of imidazole units of the co-polymer (5 mg/ml), while this concentration of free imidazole was sufficient to elute only weakly bound proteins. Complete elution of (His)6-LDH by the free imidazole was achieved only at concentrations as high as 160 mM. Thus, it was clearly demonstrated, that the efficiency of low-molecular-mass displacer could be improved significantly by converting it into a polymeric displacer having interacting groups of the same chemical nature.
使用乙烯基咪唑和乙烯基己内酰胺的合成共聚物[聚(VI - VCL)]作为置换剂，以置换模式成功地在Cu2 + -亚氨基二乙酸琼脂糖上进行了蛋白质的固定化金属亲和色谱(IMAC)。乙烯基己内酰胺使该共聚物具有热敏性，例如，当温度升高到48℃时，共聚物几乎能从水溶液中定量沉淀的性质。使用IMAC柱纯化了用(His)6标签修饰的嗜热脂肪芽孢杆菌的热稳定乳酸脱氢酶[(His)6 - LDH]。首次清楚地证明，与具有相同化学性质的单体置换剂(咪唑)相比，聚合物置换剂[聚(VI - VCL)]更有效，这可能是由于同一大分子内的咪唑基团与一个Cu2 +离子的多点相互作用。在共聚物咪唑单元浓度为3.7 mM(5 mg/ml)时，已实现结合的(His)6 - LDH的完全洗脱，而该浓度的游离咪唑仅足以洗脱弱结合的蛋白质。只有在高达160 mM的浓度下，游离咪唑才能实现(His)6 - LDH的完全洗脱。因此，清楚地证明，通过将低分子量置换剂转化为具有相同化学性质的相互作用基团的聚合物置换剂，可以显著提高其效率。
