Chapter title |
Cloning of Single-Chain Antibody Variants by Overlap-Extension PCR for Evaluation of Antibody Expression in Transient Gene Expression
|
---|---|
Chapter number | 4 |
Book title |
Heterologous Protein Production in CHO Cells
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6972-2_4 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6971-5, 978-1-4939-6972-2
|
Authors |
Patrick Mayrhofer, Renate Kunert |
Editors |
Paula Meleady |
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. |
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