A general platform for efficient extracellular expression and purification of Fab from Escherichia coli
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Antigen-binding fragments (Fabs) are an important part of monoclonal antibody (mAb) therapeutics and can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, Fabs tend to form undesirable aggregates when expressed in the cytoplasm of E. coli, substantially reducing the yield of correctly folded proteins. To solve this problem, in this study, we used five Fab fragments targeting IGF1R, Her2, VEGF, RANKL, and PD-1 to develop a novel system employing the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence to facilitate the efficient expression and extracellular secretion of Fabs. Following phosphate starvation, all five Fab fragments were expressed in BL21(DE3), were largely secreted into the culture medium, and then, were further purified by affinity chromatography specific to the constant region of the light chain. The purified Fab products were evaluated and were found to have high purity, antigen-binding affinity, and in vitro bioactivity. The mechanism experiments revealed that (1) BL21(DE3) had significantly higher productivity than the K-12 strains investigated; (2) the secretion ability of the PhoA promoter was superior to that of the T7 promoter; and (3) signal peptide, STII, showed higher extracellular secretion efficiency than pelB. Our findings strongly suggested that the phoA-STII-facilitated extracellular production platform is highly promising for application in the manufacturing of Fab fragments for both academic and industrial purposes.
KeywordsFab phoA STII Extracellular production E. coli
This work was supported in part by the National Natural Science Foundation of China (No. 81773621 to Zhu J.) and the Science and Technology Commission of Shanghai Municipality (No. 17431904500 & 17ZR1413700 to Lu H.).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animal experiments.
- Akiyama Y, Nonomura C, Kondou R, Miyata H, Ashizawa T, Maeda C, Mitsuya K, Hayashi N, Nakasu Y, Yamaguchi K (2016) Immunological effects of the anti-programmed death-1 antibody on human peripheral blood mononuclear cells. Int J Oncol 49(3):1099–1107. https://doi.org/10.3892/ijo.2016.3586 CrossRefGoogle Scholar
- Chen HH, Li NH, Xie YQ, Jiang H, Yang XY, Cagliero C, Shi SW, Zhu CC, Luo H, Chen JS, Zhang L, Zhao ML, Feng L, Lu HL, Zhu JW (2017) Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli. Appl Microbiol Biotechnol 101(13):5267–5278. https://doi.org/10.1007/s00253-017-8265-x CrossRefGoogle Scholar
- Douthwaite JA, Finch DK, Mustelin T, Wilkinson TC (2017) Development of therapeutic antibodies to G protein-coupled receptors and ion channels: opportunities, challenges and their therapeutic potential in respiratory diseases. Pharmacol Ther 169:113–123. https://doi.org/10.1016/j.pharmthera.2016.04.013 CrossRefGoogle Scholar
- Humphreys DP, Carrington B, Bowering LC, Ganesh R, Sehdev M, Smith BJ, King LM, Reeks DG, Lawson A, Popplewell AG (2002) A plasmid system for optimization of Fab' production in Escherichia coli: importance of balance of heavy chain and light chain synthesis. Protein Expr Purif 26(2):309–320CrossRefGoogle Scholar
- Ji WW, Yu DA, Yang P, Fang P, Cao YX, Li H, Xie N, Yan SS (2017) Recombinant humanized anti-vascular endothelial growth factor monoclonal antibody efficiently suppresses laser-induced choroidal neovascularization in rhesus monkeys. Eur J Pharm Sci 109:624–630. https://doi.org/10.1016/j.ejps.2017.09.021 CrossRefGoogle Scholar
- Kim SJ, Ha GS, Lee G, Lim SI, Lee CM, Yang YH, Lee J, Kim JE, Lee JH, Shin Y, Kim CW, Lee DE (2018) Enhanced expression of soluble antibody fragments by low-temperature and overdosing with a nitrogen source. Enzym Microb Technol 115:9–15. https://doi.org/10.1016/j.enzmictec.2018.04.002 CrossRefGoogle Scholar
- Lowe J, Araujo J, Yang J, Reich M, Oldendorp A, Shiu V, Quarmby V, Lowman H, Lien S, Gaudreault J, Maia M (2007) Ranibizumab inhibits multiple forms of biologically active vascular endothelial growth factor in vitro and in vivo. Exp Eye Res 85(4):425–430. https://doi.org/10.1016/j.exer.2007.05.008 CrossRefGoogle Scholar
- Karwowski W, Lekesiz K, Koc-Żórawska E, Wnuczko K, Borysewicz-Sanczyk H, Naumnik B (2017) Effects of 17β-estradioland raloxifene on endothelial OPG and RANKL secretion. Ginekol Pol 88(4):167-173. https://doi.org/10.5603/GP.a2017.0033
- Papadopoulos N, Martin J, Ruan Q, Rafique A, Rosconi MP, Shi EG, Pyles EA, Yancopoulos GD, Stahl N, Wiegand SJ (2012) Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF trap, ranibizumab and bevacizumab. Angiogenesis 15(2):171–185. https://doi.org/10.1007/s10456-011-9249-6 CrossRefGoogle Scholar
- Rezaie F, Davami F, Mansouri K, Agha Amiri S, Fazel R, Mahdian R, Davoudi N, Enayati S, Azizi M, Khalaj V (2017) Cytosolic expression of functional fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase(R) cultivation mode. J Appl Microbiol 123:134–144. https://doi.org/10.1111/jam.13483 CrossRefGoogle Scholar
- Skalniak L, Zak KM, Guzik K, Magiera K, Musielak B, Pachota M, Szelazek B, Kocik J, Grudnik P, Tomala M, Krzanik S, Pyrc K, Domling A, Dubin G, Holak TA (2017) Small-molecule inhibitors of PD-1/PD-L1 immune checkpoint alleviate the PD-L1-induced exhaustion of T-cells. Oncotarget 8(42):72167–72181. https://doi.org/10.18632/oncotarget.20050 CrossRefGoogle Scholar
- Willard D, Chen WJ, Barrett G, Blackburn K, Bynum J, Consler T, Hoffman C, Horne E, Iannone MA, Kadwell S, Parham J, Ellis B (2000) Expression, purification, and characterization of the human receptor activator of NF-kappaB ligand (RANKL) extracellular domain. Protein Expr Purif 20(1):48–57. https://doi.org/10.1006/prep.2000.1278 CrossRefGoogle Scholar