Abstract
Single-chain fragment variable–fragment crystallizable antibody constructs (scFv-Fc) are homodimeric proteins representing valuable alternatives to heterotetrameric full-length IgG molecules to study protein properties and product-dependent cellular behavior. In contrast to naturally occurring antibodies, these artificial molecules are assembled from functional antibody domains to reduce molecule complexity and enhance antibody expression levels. The scFv-Fc format retains critical antibody functions such as antigen binding affinity and antibody effector functions. Here, we present a protocol to convert the full-length anti-HIV-1 IgG1 antibody 2F5 into a scFv-Fc construct. Variable and constant regions are amplified by conventional PCR reactions and assembled by a single overlap-extension PCR reaction. The amplified product is then cloned into a mammalian expression vector suitable for high-titer transient gene expression. This workflow can be applied to any antibody sequence by adapting the specific primer sequences to the antibody of choice.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Aggarwal S (2014) What’s fueling the biotech engine-2012 to 2013. Nat Biotechnol 32:32–39
Hansel TT, Kropshofer H, Singer T, Mitchell JA, George AJT (2010) The safety and side effects of monoclonal antibodies. Nat Rev Drug Discov 9:325–338
Beck A, Wurch T, Bailly C, Corvaia N (2010) Strategies and challenges for the next generation of therapeutic antibodies. Nat Rev Immunol 10:345–352
Carter PJ (2006) Potent antibody therapeutics by design. Nat Rev Immunol 6:343–357
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Mayrhofer, P., Kunert, R. (2017). Cloning of Single-Chain Antibody Variants by Overlap-Extension PCR for Evaluation of Antibody Expression in Transient Gene Expression. In: Meleady, P. (eds) Heterologous Protein Production in CHO Cells. Methods in Molecular Biology, vol 1603. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6972-2_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6972-2_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6971-5
Online ISBN: 978-1-4939-6972-2
eBook Packages: Springer Protocols