Abstract
The quality of the target antigen is very important in order to generate a good antibody, in particular when binding to a conformational epitope is desired. The use of mammalian cells for recombinant protein expression provides an efficient machinery for the correct folding and posttranslational modification of proteins. In this chapter, we describe a process to rapidly generate semi-stable human cell lines secreting a recombinant protein of interest into the culture medium. Simple disposable bioreactors that can be used in any standard cell culture laboratory enable the production of recombinant protein in the multi-milligram range. The protein can be readily purified from the culture supernatant by immobilized metal affinity chromatography. In addition, by inserting a tag recognized by a co-expressed biotin ligase, the protein can be biotinylated during the secretion process. This greatly facilitates the immobilization of the protein for assay development or for antibody isolation using in vitro selection technologies.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Takatsuka S, Sekiguchi A, Tokunaga M, Fujimoto A, Chiba J (2011) Generation of a panel of monoclonal antibodies against atypical chemokine receptor CCX-CKR by DNA immunization. J Pharmacol Toxicol Methods 63:250–257
Zhao Q, Zhu J, Zhu W, Li X, Tao Y, Lv X, Wang X, Yin J, He C, Ren X (2013) A monoclonal antibody against transmissible gastroenteritis virus generated via immunization of a DNA plasmid bearing TGEV S1 gene. Monoclon Antib Immunodiagn Immunother 32:50–54
Roivainen M, Alakulppi N, Ylipaasto P, Eskelinen M, Paananen A, Airaksinen A, Hovi T (2005) A whole cell immunization-derived monoclonal antibody that protects cells from coxsackievirus A9 infection binds to both cell surface and virions. J Virol Methods 130:108–116
Brooks SA (2006) Protein glycosylation in diverse cell systems: implications for modification and analysis of recombinant proteins. Expert Rev Proteomics 3:345–359
Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17:353–358
Li W, Gao M, Liu W, Kong Y, Tian H, Yao W, Gao X (2012) Optimized soluble expression and purification of an aggregation-prone protein by fusion tag systems and on-column cleavage in Escherichia coli. Protein Pept, Lett
Peroutka Iii RJ, Orcutt SJ, Strickler JE, Butt TR (2011) SUMO fusion technology for enhanced protein expression and purification in prokaryotes and eukaryotes. Methods Mol Biol 705:15–30
Qi Y, Zou Z, Zou H, Fan Y, Zhang C (2011) [Intrinsic prokaryotic promoter activity of SUMO gene and its applications in the protein expression system of Escherichia coli. Sheng Wu Gong Cheng Xue Bao 27:952–962
Sun P, Tropea JE, Waugh DS (2011) Enhancing the solubility of recombinant proteins in Escherichia coli by using hexahistidine-tagged maltose-binding protein as a fusion partner. Methods Mol Biol 705:259–274
Tirat A, Freuler F, Stettler T, Mayr LM, Leder L (2006) Evaluation of two novel tag-based labelling technologies for site-specific modification of proteins. Int J Biol Macromol 39:66–76
Magistrelli G, Malinge P, Lissilaa R, Fagete S, Guilhot F, Moine V, Buatois V, Delneste Y, Kellenberger S, Gueneau F, Ravn U, Kosco-Vilbois M, Fischer N (2010) Rapid, simple and high yield production of recombinant proteins in mammalian cells using a versatile episomal system. Protein Expr Purif 72:209–216
Sung K, Maloney MT, Yang J, Wu C (2011) A novel method for producing mono-biotinylated, biologically active neurotrophic factors: an essential reagent for single molecule study of axonal transport. J Neurosci Methods 200:121–128
Magistrelli G, Malinge P, Anceriz N, Desmurs M, Venet S, Calloud S, Daubeuf B, Kosco-Vilbois M, Fischer N (2012) Robust recombinant FcRn production in mammalian cells enabling oriented immobilization for IgG binding studies. J Immunol Methods 375:20–29
Bruce MP, Boyd V, Duch C, White JR (2002) Dialysis-based bioreactor systems for the production of monoclonal antibodies–alternatives to ascites production in mice. J Immunol Methods 264:59–68
Kimura A, Adachi N, Horikoshi M (2003) Series of vectors to evaluate the position and the order of two different affinity tags for purification of protein complexes. Anal Biochem 314:253–259
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Magistrelli, G., Malinge, P. (2014). Antigen Production for Monoclonal Antibody Generation. In: Ossipow, V., Fischer, N. (eds) Monoclonal Antibodies. Methods in Molecular Biology, vol 1131. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-992-5_1
Download citation
DOI: https://doi.org/10.1007/978-1-62703-992-5_1
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-991-8
Online ISBN: 978-1-62703-992-5
eBook Packages: Springer Protocols