Publications

Abstract

Single-domain antibodies represent an emerging class of antibody fragments with promising therapeutic and diagnostic potential. As a result, multiple strategies have been developed in order to improve their biophysical and/or biological properties. In particular, the fusion of single-domain antibodies to the Fc part of an IgG molecule has become a common protein engineering approach toward this aim. Here, we describe a detailed protocol for a streamlined laboratory-scale production of VH single-domain antibodies as Fc fusions in mammalian cells. Firstly, DNA sequence encoding VH domain of interest fused to an IgG Fc is synthesized as a double-stranded gene fragment. Secondly, the DNA fragment is directly assembled into a restriction enzyme-digested vector in an assembly reaction. Finally, vector carrying the VH-Fc-fusion construct is introduced into suspension-adapted mammalian cells for transient expression of the Fc chimeric fusion. One-week post-transfection, the expressed Fc-fusion protein is purified using protein A/G affinity chromatography. Using this protocol, we were able to clone, express, and purify milligrams of isolated anti-HER2 VH domain as a mouse IgG2c Fc fusion in less than 2 weeks. This protocol can be readily modified to express proteins of interest other than VH domains as Fc fusions.