Luksa J, Menart V, Milicic S, Kus B, Gaberc-Porekar V, Josic D
Lek, Pharmaceutical and Chemical Company, Research and Development, Ljubljana Slovenia.
J Chromatogr A. 1994 Feb 11;661(1-2):161-8. doi: 10.1016/0021-9673(94)85186-7.
The recombinant human tumour necrosis factor alpha from an extract of Escherichia coli was enriched to homogeneity according to specific activity and sodium dodecyl sulphate-polyacrylamide gel electrophoresis by purification using anion-exchange HPLC and hydrophobic interaction HPLC. Parallel experiments with the same separation methods, but carried out with membrane chromatography on compact discs, gave similar results in terms of yield and purity of the product. The active form of the protein is a trimer. The second isolation step, hydrophobic interaction chromatography, causes dissociation of the trimer into monomers and a partial loss of the biological activity of the protein. The phenomenon occurs on both the column and the disc. This in turn indicates strongly that the dissociation of the protein is a consequence of interaction between the samples and the hydrophobic ligand, and is not caused by non-specific interaction with the matrix.
从大肠杆菌提取物中获得的重组人肿瘤坏死因子α，通过阴离子交换高效液相色谱法和疏水相互作用高效液相色谱法进行纯化，根据比活性和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，将其富集至同质。使用相同的分离方法，但在光盘上进行膜色谱法的平行实验，在产物的产率和纯度方面得到了相似的结果。该蛋白质的活性形式是三聚体。第二个分离步骤，即疏水相互作用色谱法，会导致三聚体解离成单体，并使蛋白质的生物活性部分丧失。这种现象在柱上和光盘上都会发生。这进而有力地表明，蛋白质的解离是样品与疏水配体之间相互作用的结果，而不是与基质的非特异性相互作用所导致的。
