Haelewyn J, De Ley M
Laboratory of Biochemistry, Katholieke Universiteit Leuven, Belgium.
Biochem Mol Biol Int. 1995 Dec;37(6):1163-71.
A fast purification method for recombinant human interferon-gamma, produced in E. coli, was elaborated. Human IFN-gamma accumulated in the cytoplasm of E. coli cells as inclusion bodies (IB). After lysis, the IB were isolated from the cell debris by means of a density gradient ultracentrifugation, and solubilized in 6 M guanidine hydrochloride. The subsequent refolding step was optimized for a maximal recovery of the biologically active dimer. Refolded IFN-gamma was then purified to homogeneity in a single cation exchange chromatographic step, yielding 12.5 mg protein per liter E. coli culture. The dimeric nature of the refolded protein was visualized by means of interchain cross-linking. In a subsequent Western blot the resulting derivative was recognized by a panel of five monoclonal antibodies, indicating that those epitopes on the protein surface remained unaffected upon cross-linking.
阐述了一种用于纯化在大肠杆菌中产生的重组人干扰素-γ的快速方法。人干扰素-γ以包涵体（IB）的形式积累在大肠杆菌细胞的细胞质中。裂解后，通过密度梯度超速离心从细胞碎片中分离出包涵体，并将其溶解在6 M盐酸胍中。随后的复性步骤针对生物活性二聚体的最大回收率进行了优化。然后通过单步阳离子交换色谱将复性的干扰素-γ纯化至同质，每升大肠杆菌培养物产生12.5 mg蛋白质。通过链间交联观察到复性蛋白的二聚体性质。在随后的蛋白质印迹中，所得衍生物被一组五种单克隆抗体识别，表明蛋白质表面的那些表位在交联后未受影响。
