Fluorescent Citrine-IgG fusion proteins produced in mammalian cells.
Genetically encoded fluorescent antibodies are desirable for many applications in biotechnology, proteomics, microscopy, cell biology and molecular diagnostics, although efficient production of fluorescent IgGs in mammalian cells has been hampered by different and mutually incompatible secretion- and folding-requirements of antibodies and green fluorescent protein-derived fluorescent entities. Here, we show that this hurdle can be overcome by generating whole antibody fusions with Citrine, a modified yellow fluorescent protein that folds properly in the endoplasmic reticulum of mammalian cells. Applying optimized connector sequences, one or more Citrine molecules can be fused to different positions of IgGs without interfering with folding, secretion or function of the fusion proteins. These proteins can be transiently expressed and purified to similar yields as unmodified antibodies using standard technologies. IgG-Citrine fusions fully retain binding specificity and affinity, and can be applied to assays that require labeled IgG. A particularly interesting feature is the pH-dependency of Citrine fluorescence. This makes IgG-Citrine fusion proteins a valuable tool to track antibody target binding, internalization and subsequent intracellular trafficking to acidic compartments.
Haas
Alexander K
AK
Roche Diagnostics GmbH, Penzberg, Federal Republic of Germany. alexander.haas@roche.com
von Schwerin
Christoffer
C
Matscheko
Daniela
D
Brinkmann
Ulrich
U
D000915:Antibody Affinity; D001426:Bacterial Proteins / Q000378:metabolism*; D002460:Cell Line; D002469:Cell Separation; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D007074:Immunoglobulin G; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C434479:citrine protein, bacteria