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Expression of Human VH Single Domains as Fc Fusions in Mammalian Cells


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.

Type Book section
ISBN 1940-6029 (Electronic) 1064-3745 (Linking)
Authors Abdelatti, M.; Schofield, P.; Christ, D.
Responsible Garvan Author Prof Daniel Christ
Publisher Name Methods in Molecular Biology
Published Date 2019-03-01
Published Volume 1953
Published Pages 121-136
Status Always Electronic
DOI 10.1007/978-1-4939-9145-7_8
URL link to publisher's version