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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2016, Vol. 36 Issue (9): 31-37    DOI: 10.13523/j.cb.20160904
技术与方法     
大肠杆菌可溶性表达抗TNF-α Fab的工艺优化
张宇萌1,2, 童梅2, 陆小冬2, 米月1, 莫婷1,2, 刘金毅2, 姚文兵1
1 中国药科大学生命科学与技术学院 南京 210009;
2 北京三元基因药业股份有限公司 北京 102600
Expression of Soluble Anti-TNF-α Fab in E.coli: Optimization for Technological Process
ZHANG Yu-meng1,2, TONG Mei2, LU Xiao-dong2, MI Yue1, MO Ting1,2, LIU Jin-yi2, YAO Wen-bing1
1 Institute of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China;
2 Beijing Tri-Prime Gene Pharmaceutical Co. Ltd., Beijing 102600, China
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摘要：

目的：通过构建抗TNF-α Fab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。
关键词： Fab;  Protein L;  大肠杆菌;  DOE;  溶液萃取;  TNF-α  疏水作用层析    
Abstract:

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.
Key words: HIC    E.coli    TNF-α    Fab    DOE    Protein L    Extraction
收稿日期: 2016-03-09 出版日期: 2016-09-25
ZTFLH:  Q816  
基金资助:

北京市科技计划资助项目（Z141100000514008）
通讯作者: 刘金毅, 姚文兵     E-mail: liujinyi@triprime.com;wbyao@cpu.edu.cn
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引用本文:

张宇萌, 童梅, 陆小冬, 米月, 莫婷, 刘金毅, 姚文兵. 大肠杆菌可溶性表达抗TNF-α Fab的工艺优化[J]. 中国生物工程杂志, 2016, 36(9): 31-37.

ZHANG Yu-meng, TONG Mei, LU Xiao-dong, MI Yue, MO Ting, LIU Jin-yi, YAO Wen-bing. Expression of Soluble Anti-TNF-α Fab in E.coli: Optimization for Technological Process. China Biotechnology, 2016, 36(9): 31-37.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160904    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I9/31

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