Second Clinical College, Dalian Medical University, Dalian, China.
Appl Biochem Biotechnol. 2009 Dec;159(3):634-41. doi: 10.1007/s12010-008-8495-6. Epub 2009 Jan 25.
Efficient refolding of recombinant proteins in the forms of inclusion bodies at higher concentration remains challenging. Here, we report a strategy of a dual-gradient hydrophobic interaction chromatography (HIC) mode to refold recombinant human granulocyte colony-stimulating factor from its inclusion bodies at high protein concentration. The strategy was taken to meet the demand of dynamic refolding proceeding by gradually decrease the denaturant (guanidine-HCl) concentration and gradually increase the hydrophilicity of media (column of Poros PE 20) with glycerol as additive to provide a mild refolding surroundings. Compared with dilution method, this dual-gradient HIC process gave about 8.5-fold of increase in specific activity and 30% increase in soluble protein recovery. Furthermore, much higher protein concentration could be obtained at the same time.
在较高浓度下以包涵体形式高效复性重组蛋白仍然具有挑战性。在这里，我们报告了一种双重梯度疏水相互作用色谱（HIC）模式的策略，用于从高浓度的包涵体中复性重组人粒细胞集落刺激因子。该策略旨在通过逐渐降低变性剂（盐酸胍）浓度和逐渐增加亲水性介质（甘油作为添加剂的 Poros PE 20 柱）来满足动态复性过程的需求，从而提供温和的复性环境。与稀释法相比，这种双重梯度 HIC 过程使比活度提高了约 8.5 倍，可溶性蛋白回收率提高了 30%。此外，同时可以获得更高的蛋白质浓度。
