
大肠杆菌可溶性表达抗TNF-α Fab的工艺优化
张宇萌, 童梅, 陆小冬, 米月, 莫婷, 刘金毅, 姚文兵
中国生物工程杂志 ›› 2016, Vol. 36 ›› Issue (9) : 31-37.
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-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。
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.
Fab / Protein L / 大肠杆菌 / DOE / 溶液萃取 / TNF-&alpha / 疏水作用层析 {{custom_keyword}} /
HIC / E.coli / TNF-α / Fab / DOE / Protein L / Extraction {{custom_keyword}} /
[1] Tanaka Y. Current concepts in the management of rheumatoid arthritis. Korean J Intern Med, 2016, 31(2):210-218.
[2] Tanaka Y. Intensive treatment and treatment holiday of TNF-inhibitors in rheumatoid arthritis. Curr Opin Rheumatol, 2012, 24(3):319-326.
[3] Makrides S C. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiology, 1996, 60(3):512-538.
[4] Olins P O, Lee S C. Recent advances in heterologous gene expression in Escherichia coli. Curr Opin Biotechnol, 1993, 4(5):520-525.
[5] 夏金兰,王晶,张倩,等. 嗜酸氧化亚铁硫杆菌ATCC23270周质蛋白的选择性提取及差异表达. 中南大学学报(自然科学版), 2009, 40(4):845-850. Xia J L, Wang J, Zhang Q, et al. Selective extraction and differential electrophoregrams analysis of periplasmic proteins of Acidithiobacillus ferrooxidans ATCC23270. Journal of Central South University(Science and Technology), 2009, 40(4):845-850.
[6] Kang H J, Kim H J, Cha S H, et al. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life. Immunology Letters, 2016, 169:33-40.
[7] Lakhrif Z, Pugnière M, Henriquet C, et al. A method to confer protein L binding ability to any antibody fragment. MAbs, 2016, 8(2):379-388.
[8] Graille M, Stura E A, Housden N G, et al. Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins. Structure, 2001, 9(8):679-687.
[9] 王清,蒋葵,李俊,等. 抗人大肠癌P-gp Fab抗体的制备、纯化及初步鉴定. 生物技术, 2013, 23(3):65-70. Wang Q, Jiang K, Li J, et al. Production,purification and identification of the Fab antibody against human colorectal cancer P-gp. Biotechnology, 2013, 23(3):65-70.
[10] Mayolo-Deloisa K, Lienqueo M E, Andrews B, et al. Hydrophobic interaction chromatography for purification of monoPEGylated RNase A. J Chromatogr A, 2012, 1242:11-1716.
[11] Mirani M R, Rahimpour F. Thermodynamic modelling of hydrophobic interaction chromatography of biomolecules in the presence of salt. J Chromatogr A, 2015, 1422:170-177.
[12] Murphy P J, Stone O J, Anderson M E. Automated hydrophobic interaction chromatography column selection for use in protein purification. J Vis Exp, 2011, 55(21):3060-3065.
[13] Arakawa T, Kita Y, Ejima D, et al. Solvent modulation of column chromatography. Protein Pept Lett, 2008, 15(6):544-555.
[14] Faust G, Janzen N H, Bendig C, et al. Feeding strategies enhance high cell density cultivation and protein expression in milliliter scale bioreactors. Biotechnology, 2014, 9(10):1293-1303.
[15] Velmurugan N, Kim H S, Jeong K J, et al. Enhanced production of human FccRⅡa receptor by high cell density cultivation of Escherichia coli. Protein Expression and Purification, 2011, 79(1):60-65.
北京市科技计划资助项目(Z141100000514008)
/
〈 |
|
〉 |