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目的 在成功构建人粒/巨噬细胞集落刺激因子(GM—CSF)高效表达克隆的基础上对其表达产物进行分离纯化,并对rhGM—CSF在溶液中的构象、稳定性、变复性等进行研究。方法 在纯化研究基础上利用傅里叶变换红外光谱技术(FTIR)对rhGM—CSF的构象进行研究,同时应用聚乙二醇(PEG)和牛肝二硫键异构酶(PDI)对GM—CSF的复性及体外折叠作用进行分析。结果 利用FTIR技术可见GM—CSF在D_2O溶液中20℃时含35％0α—螺旋,32％β—螺旋,11％转角和20％无规卷曲。适当PEG可使蛋白得率和比活性均达到最高,PDI可以提高蛋白质的正确折叠率。结论 采用FTIR研究蛋白质在溶液中的构象耗时短、重复性好、样品耗量小,对产品的质量控制及批间产品的稳定性具有指导价值。加入适当比例的PEG和PDI可使蛋白得率和比活性明显提高。
OBJECTIVE To isolate and purify the expressed product of human granulocyte / macrophage colony stimulating factor (GM-CSF) efficiently and to study the conformation, stability and refolding of rhGM-CSF in solution research. Methods The conformation of rhGM-CSF was studied by Fourier transform infrared spectroscopy (FTIR) on the basis of purification. At the same time, the effects of PEG and bovine liver disulfide isomerase (PDI) on the conformation of GM-CSF Refolding and in vitro folding analysis. Results The results of FTIR showed GM-CSF contained 35% 0α-helix, 32% β-helix, 11% angle and 20% random coil at 20 ℃ in D 2 O solution. Appropriate PEG can achieve the highest protein yield and specific activity, PDI can improve the correct protein folding rate. Conclusion The FTIR study of protein conformation in solution has the advantages of short time-consuming, good repeatability and low sample consumption, which is of guiding value for product quality control and inter-batch product stability. Addition of appropriate ratios of PEG and PDI can significantly improve the protein yield and specific activity.