Abstract
Antibody-fusion proteins with ligands, e.g., of the TNF superfamily (TNFSF) can be adequately produced in mammalian expression systems. Here, we describe the transient production in adherent and suspension human embryonic kidney cells at laboratory scale, followed by purification procedures applying protein A and immobilized metal affinity chromatography for proteins with Fc domain and 6 × histidine-tag, respectively. In addition, characterization of the purified proteins by size exclusion chromatography is described.
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
Kontermann RE (2012) Antibody-cytokine fusion proteins. Arch Biochem Biophys 526(2):194–205
Müller D (2015) Antibody fusions with immunomodulatory proteins for cancer therapy. Pharmacol Ther 154:57–66
Jäger V, Büssow K, Wagner A et al (2013) High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC Biotechnol 13:52
Durocher Y, Perret S, Kamen A (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30(2):E9
Huang C (2009) Receptor-fc fusion therapeutics, traps, and MIMETIBODY technology. Curr Opin Biotechnol 20(6):692–699
Soleimanpour S, Hassannia T, Motiee M et al (2017) Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties. Crit Rev Biotechnol 37(3):371–392
Hober S, Nord K, Linhult M (2007) Protein A chromatography for antibody purification. J Chromatogr B Analyt Technol Biomed Life Sci 848(1):40–47
Kosobokova EN, Skrypnik KA, Kosorukov VS (2016) Overview of fusion tags for recombinant proteins. Biochemistry (Mosc) 81(3):187–200
Khan F, Legler PM, Mease RM et al (2012) Histidine affinity tags affect MSP1(42) structural stability and immunodominance in mice. Biotechnol J 7(1):133–147
Bremer E (2013) Targeting of the tumor necrosis factor receptor superfamily for cancer immunotherapy. ISRN Oncol 2013:371854
Fellermeier S, Beha N, Meyer JE et al (2016) Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format. Oncoimmunology 5(11):e1238540
Siegemund M, Seifert O, Zarani M et al (2016) An optimized antibody-single-chain TRAIL fusion protein for cancer therapy. MAbs 8(5):879–891
Hutt M, Marquardt L, Seifert O et al (2017) Superior properties of Fc-comprising scTRAIL fusion proteins. Mol Cancer Ther 16(12):2792–2802
Tom R, Bisson L, Durocher Y (2008) Transfection of HEK293-EBNA1 cells in suspension with linear PEI for production of recombinant proteins. CSH Protoc 2008:pdb.prot4977. https://doi.org/10.1101/pdb.prot4977
Zhang J, Liu X, Bell A et al (2009) Transient expression and purification of chimeric heavy chain antibodies. Protein Expr Purif 65(1):77–82
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Siegemund, M., Beha, N., Müller, D. (2018). Production, Purification, and Characterization of Antibody-TNF Superfamily Ligand Fusion Proteins. In: Nevoltris, D., Chames, P. (eds) Antibody Engineering. Methods in Molecular Biology, vol 1827. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8648-4_18
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8648-4_18
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8647-7
Online ISBN: 978-1-4939-8648-4
eBook Packages: Springer Protocols