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

  • Mahmoud Abdelatti
  • Peter Schofield
  • Daniel Christ
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1953)

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.

Key words

Human VH single domains Fc fusions Mammalian expression Protein biotinylation Bio-layer interferometry 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Mahmoud Abdelatti
    • 1
    • 2
  • Peter Schofield
    • 1
  • Daniel Christ
    • 1
    • 2
  1. 1.Immunology ProgramGarvan Institute of Medical ResearchSydneyAustralia
  2. 2.Faculty of Medicine, St. Vincent’s Clinical SchoolUniversity of New South WalesSydneyAustralia

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