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Generation of Fluorescent IgG Fusion Proteins in Mammalian Cells

  • Alexander K. Haas
  • Klaus Mayer
  • Ulrich BrinkmannEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 901)

Abstract

The generation of recombinantly produced fluorescent antibody derivatives that are derived from full-length immunoglobulin G (IgG) has until now been problematic. One major reason for that lies in different and partially incompatible secretion- and folding-requirements of antibodies and green fluorescent protein (GFP) derived fluorescent entities in mammalian cells. The use of citrine as fluorescent fusion entity can overcome this limitation. Citrine is a modified yellow fluorescent protein (YFP) derivative which in contrast to GFP and yellow fluorescent protein (YFP) folds effectively and properly in the endoplasmic reticulum (ER) of mammalian cells. Provided that proper design parameters regarding fusion positions and linker/connector sequences are applied, citrine can be fused to different positions of IgGs and be expressed without interfering with secretion capability or functionality of IgG–citrine derivatives. Because IgG–citrine fusions are stable and retain biophysical properties of IgGs, they can be expressed and purified in the same manner as regular antibodies. IgG–citrine fusions not only retain the binding properties (affinity and specificity) of antibodies but also contain Fc-regions (useful for immunoassay applications), and are fully defined molecules (in contrast to antibody conjugates with fluorophores).

Key words

Immunoglobulin G Multifunctional antibody Citrine Antibody-fusion protein Green fluorescent protein Enhanced green fluorescent protein Yellow fluorescent protein Fluorobodies Fluorescent antibodies 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Alexander K. Haas
    • 1
  • Klaus Mayer
    • 1
  • Ulrich Brinkmann
    • 1
    Email author
  1. 1.Large Molecule Research, Roche Pharma Research and Early DevelopmentPenzbergGermany

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