Chen Jing, Liu Yongdong, Li Xiunan, Wang Yinjue, Ding Hong, Ma Guanghui, Su Zhiguo
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Science, Beijing 100190, China.
Protein Expr Purif. 2009 Jul;66(1):82-90. doi: 10.1016/j.pep.2009.02.004. Epub 2009 Feb 20.
The use of low concentrations of urea, guanidinium chloride or arginine has been reported in the literature to increase protein refolding and yield of active proteins by suppressing aggregate formation. However, no studies have yet examined whether these substances can exert synergistic or cooperative effects when used in combination. In this work, a comparative study was carried out on refolding of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in the presence of different concentrations of urea, guanidinium chloride or arginine. All three folding aids could inhibit the formation of insoluble aggregates of rhG-CSF but with different efficacies. A low concentration of guanidinium chloride was found to denature protein, so that rhG-CSF was not fully or correctly folded even if concentration was reduced to 1M. Low concentration of urea (2M) or arginine (0.5M) did not cause rhG-CSF denaturation, but urea was unable to suppress the formation of soluble oligomers, which persisted at a level of about 30% in refolded soluble rhG-CSF. Arginine, in contrast, could inhibit formation of all soluble oligomers. Based on these phenomena, we tested rhG-CSF folding in a mixture of 2M urea and 0.5M arginine. Kinetic analysis indicated that urea aided in suppressing insoluble precipitates, while arginine prevented formation of soluble oligomers produced by hydrophobic interaction. With this combination system, the refolding yield of rhG-CSF could be increased 2-fold.
文献报道，使用低浓度的尿素、氯化胍或精氨酸可通过抑制聚集体形成来提高蛋白质复性及活性蛋白产量。然而，尚无研究探讨这些物质联合使用时是否会产生协同或协作效应。在本研究中，我们对重组人粒细胞集落刺激因子（rhG-CSF）在不同浓度的尿素、氯化胍或精氨酸存在下的复性进行了比较研究。所有这三种折叠助剂均可抑制rhG-CSF不溶性聚集体的形成，但效果不同。发现低浓度的氯化胍会使蛋白质变性，因此即使浓度降至1M，rhG-CSF也无法完全或正确折叠。低浓度的尿素（2M）或精氨酸（0.5M）不会导致rhG-CSF变性，但尿素无法抑制可溶性寡聚体的形成，在复性后的可溶性rhG-CSF中，其含量持续保持在约30%的水平。相比之下，精氨酸可抑制所有可溶性寡聚体的形成。基于这些现象，我们测试了rhG-CSF在2M尿素和0.5M精氨酸混合物中的折叠情况。动力学分析表明，尿素有助于抑制不溶性沉淀，而精氨酸可防止由疏水相互作用产生的可溶性寡聚体的形成。通过这种组合体系，rhG-CSF的复性产量可提高2倍。
