Cloning of Single-Chain Antibody Variants by Overlap-Extension PCR for Evaluation of Antibody Expression in Transient Gene Expression

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.