大肠杆菌可溶性表达抗TNF-α Fab的工艺优化

Expression of Soluble Anti-TNF-α Fab in E.coli:Optimization for Technological Process

目的:通过构建抗TNF-αFab重组表达质粒,在大肠杆菌周质空间中高效可溶性表达抗TNF-α Fab,经工艺优化以提高目的蛋白的产率。方法:菌体经发酵罐发酵后,使用萃取方法进行周质空间可溶性目的蛋白的萃取并通过试验设计(DOE)优化萃取工艺,采用Q Sepharose Fast Flow强碱性阴离子交换柱净化,Phenyl Sepharose Fast Flow疏水柱及Protein L亲和柱纯化。结果:经DOE筛选得到了相对最优的萃取条件,该纯化方法得到的目的蛋白的体积产率可达23.5mg/L,纯度大于90%,回收率大于24%,且离子交换柱净化过程有效地保护了亲和柱,提高了亲和柱的使用寿命。抗体亲和力检测证明抗TNF-α Fab具有良好的生物学活性。结论:通过萃取工艺及纯化方法的优化,为抗TNF-α Fab的进一步工艺放大及目的蛋白的性质研究奠定了基础。

Subject：The anti-TNF-α Fab recombinant plasmid to achieve an efficient soluble expression of anti-TNF-α Fab in periplasmic space of E.coli,and to improve the yield of the target protein by optimizing technological process were constructed. Method： After fermentation,extraction method was optimized for a higher Fab yield by DOE to extract the target protein of periplasm. Ion exchange chromatography（IEX） was applied to clear the extraction,and purification was achieved by hydrophobic interaction chromatography（HIC） and Protein L affinity chromatography. Results： An optimal extraction condition by DOE and the yield was up to 23.5mg/L,the overall recovery rate was above 24% and the purity was above 90%. Ion exchange chromatography provided a good protection for affinity column. The result of affinity assay indicated that the anti-TNF-α Fab had excellent bioactivities. Conclusion： A foundation for further amplification of the production by optimizing extraction and purification process have been made.