Fluorescent Citrine-IgG fusion proteins produced in mammalian cells.

Abstract:

: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.

journal_name

MAbs

journal_title

mAbs

authors

Haas AK,von Schwerin C,Matscheko D,Brinkmann U

doi

10.4161/mabs.2.6.13179

subject

Has Abstract

pub_date

2010-11-01 00:00:00

pages

648-61

issue

6

eissn

1942-0862

issn

1942-0870

pii

13179

journal_volume

2

pub_type

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