|
|
|
| The Secretory Expression of an Anti-VEGF Antibody Fab Fragment in E.coli |
| WANG Zhi-long1, WANG Ying-ming2, LIU Zheng-zhao2, LU Da-ru1, ZHU Hua-xing2 |
1. State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China;
2. Novoprotein Scientific Inc., Shanghai 201203, China |
|
|
|
Abstract Object: To construct, express, purify anti-VEGF antibody Fab fragment in E.coli, and to assay its activity.Through controlling the fermentation conditions, Fab fragment can be achieved actively and largely by secreting to the periplasmic space or extracellular medium.Methods: The co-expression plasmid pET30a-LC-HC which has the OmpA signal peptide, SD sequence and the T7 promoter before Fd chain and L chain was successfully constructed.Then the plasmid was transformed into Escherichia coil BL21(DE3).The inducing condition including medium, temperature and IPTG concentration was also optimized. Results: The best shaking fermentation condition on Escherichia coil periplasmic secretory expression: in 1L XY1 medium that including 1.5% Tryptone, 1% Yeast Extract, 0.5% Glucose, 0.15% NaCl, 0.1% NH4Cl, 0.08% MgCl2~6H2O, the strains were cultured with 10% inoculum at 37℃, and then added 0.1mM IPTG until the late logarithmic growth phase(OD600 was about 2) to induced overnight(about 16h) at 16℃. Except periplasmic expression, there is also a considerable part of Fab that secrete into the medium. Fab fragment which secrected into the periplasm was extracted by breaking periplasm of bacteria.At the same time, the culture medium were concentrated with the hollow fiber column, and further purified Fab by ProteinG affinity chromatography.At last, we got the purified Fab antibody fragments with more than 90% purity by SDS-PAGE analysis.The protein concentration is measured with Brandford detection method.The amount of purified Fab from periplasm and medium is up to 0.4mg/L. Taking the VEGF165 as the binding antigen, the half-maximal effective concentration (EC50) of the purified Fab is 30ng/ml being analyzed indirectly by ELISA, which show a high antigen-binding activity. Then 3.7L fermentor with 2L volume of XY1 culture mediun is used to do small pilot scale fermentation.The final cell yield is 30g/L in the 3.7L fermenter with 2L culture medium, and the concentration of Fab antibody purified by affinity column is 1.94mg/L. Conclusion: Fab fragment can be successful and high-efficiently secrected into the periplasmic space and extracellular medium through constructing the co-expression vector and optimizing the fermentation condition, and has highly activity against VEGF. It provides research basis for large-scale preparation of Fab antibody fragment.
|
|
Received: 12 May 2014
Published: 25 July 2014
|
|
|
|
|
|
[1] Venturi M, Seifert C, Hunte C. High level production of functional antibody Fab fragments in an oxidizing bacterial cytoplasm. J Mol Biol, 2002, 315:1-8.
[2] Leung D W,Cachianes G,Kuang W J,et al.Vascular endothelial growth factor is a secreted angiogenic mitogen.Scinece,1989,246:1306-1309
[3] Presta L G,Chen H,OConnor S J,et al.Humanization of an antivasular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders.Cancer Res,1997,57(20):4593-4599.
[4] Friedrich L, Stangl S, Hahne H, et al. Bacterial production and functional characterization of the Fab fragment of the murine IgG1/lambda monoclonal antibody cmHsp70.1, a reagent for tumour diagnostics. Protein Eng Des Sel, 2010,23(4): 161-168.
[5] 刘运宏,侯宗柳.大肠杆菌表达抗原结合片段的策略.中国组织工程研究与临床康复,2011,15(15):2821-2824. Liu Y H,Hou Z L.Strategies for fragmen antigen bingding expression in Escherichia coli.Journal of Clinical Rehabilitative Tissue Engineering Research, 2011,15(15):2821-2824.
[6] 熊冬生,郑梦杰,刘银星,等.抗CD20嵌合抗体片段F(abc)2突变体在大肠杆菌中的高效分泌表达.生物工程学报,2004.9.20(5):673-678. Xiong D SH, Zheng M J, Liu Y X,et al.High level expression of chimeric antibody fragment F(abc)2 directed against CD20 in Escherichia coli.Chinese Journal of Biotechnology, 2004.9.20(5):673-678.
[7] Brandon L, Vikram S, Vaughn Smider. Co-expression of antibody fab heavy and light chain genes from separate evolved compatible replicons in E. coli. Immunol Methods, 2006,317(1-2): 56-63.
[8] Levy R, Weiss R, Chen G, et al. Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones. Protein Expr Purif, 2001;23(2):338-347.
[9] David P. Humphreysa N W, Alastair L, et al. Co-expression of human protein disulphide isomerase (PDI) can increase the yield of an antibody Fab' fragment expressed in Escherichia coli. FEBS Letters,1996,380: 194-197.
[10] Winter J,Neubauer P. Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA. J Biotechnol,2000,84:175.
[11] Choi J H, Lee S Y. Secretory and extracellular production of recombinant proteins using Escherichia coli.Appl Microbiol Biotechno,2004, 64(5) 625-635.
[12] Shokri A,Sande’n A M,Larsson G.Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli.Appl Microbiol Biotechnol,2003,60(6):654-664.
[13] Balderas Hernandez, V. E.Paz Maldonado,et al. Periplasmic expression and recovery of human interferon gamma in Escherichia coli. Protein Expr Purif, 2008,59(1): 169-174.
[14] Mergulhao, F. J.Summers, D. K.Monteiro, G. A. Recombinant protein secretion in Escherichia coli. Biotechnol Advances, 2005,23(3): 177-202.
[15] Donovan R S, Robinson C W, Glick B R. Review: Optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter. Journal of Industrial Microbiology, 1996,16(3): 145-154.
[16] Yan Ge1, Yongjing Chen, Songguang Ju, Xue-Guang Zhang. Functional expression of chimeric Fab of an anti-CD40L mAb: Vector design and culture condition optimization. Biomed Pharmacother, 2011,65(1): 52-59
[17] Corisdeo S, Wang B. Functional expression and display of an antibody Fab fragment in Escherichia coli: study of vector designs and culture conditions.Protein Expr Purif, 2004,34(2): 270-279.
[18] Rodríguez-Carmona E, et al. Recombinant Fab expression and secretion in Escherichia coli continuous culture at medium cell densities: Influence of temperature.Process.Biochemistry, 2012,47(3): 446-452.
[19] 李时荣,王琰,孙勤暖,等.抗胃癌鼠单抗Fab段在大肠杆菌中的表达.内蒙古医学院学报.2006,28(6):503-505. Li Sh R,Wang Y,Sun Q N,et al.Expression of monoclonal mouse anti-Fab frangment in E.coli. Acta Academiae Medicinae Neimongol, 2006,28(6):503-505.
[20] Kaisa Ukkonen J V, Antti Vasala, Peter Neubauer. Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E.coli cultures. Microbial Cell Factories, 2013,12:73
[21] 邓宁,陈文吟,向军俭,等.亲和层析纯化重组人源性抗HBsAg Fab抗体.细胞与分子免疫学杂志,2004,20(5):575-577. Deng N,Chen W Y,Xiang J J,et al. Purification of recombinant humanized anti-HBsAg Fab antibody by affinity chromatography. Chinese Journal of Cellular and Molecular Immunology,2004, 20(5):575-577.
[22] 饶桂荣,陈文吟,粟宽源,等.重组人抗HBs-Fab抗体的纯化及结构分析.中国生物制品学杂志,2006,19(1):54-57. Rao G R,Chen W Y,Su K Y,et al. Purification and analysis of structure of Fab fragement of recombinant human anti-HBs.Chin J Biologicals January,2006,19(1):54-57.
[23] 邓宁,向军俭,饶桂荣,等.重组人源性抗HBsAg-Fab抗体纯化方法的比较研究.生物工程学报,2004,20(6):884-889. Deng N,Xiang J J,Rao G R, et al. Study on the Methods of Purification of Recombinant Humanized anti-HBsAg Fab Antibody. Chinese Journal of Biotechnology,2004,20(6):884-889.
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
