Abstract
Aim:
To develop a simple method for monitoring protein localization of epidermal growth factor (EGF) in living cells.
Methods:
Enhanced green fluorescent protein (EGFP) was used as an autofluorescent tag to label EGF ligands. SDS-PAGE and Western blot analysis were used to detect the expression of the EGFP-tagged EGF (EGFP-EGF) protein. The cell-binding and internalization activity of EGFP-EGF were analyzed by fluorescence-activated cell sorting (FACS) and confocal microscopy.
Results:
EGFP-EGF protein was expressed in Escherichia coli and purified. A cell-binding assay demonstrated that the EGFP-EGF protein could bind efficiently to the cells expressing EGFR. The binding and internalization of EGFP-EGF can be visualized even at a very low concentration under confocal microscopy. The FACS-based assay for internalization activity indicated the accumulation of internalized EGFP-EGF over time. Furthermore, the results of the competition assay indicated its EGFR binding specificity. Using such a method, it does not need to label EGF with chemicals and avoid light in the experimental process.
Conclusion:
The fusion protein EGFP-EGF has several characters including high sensitivity, stability and convenience for manipulation, and is a powerful tool for the study of EGF endocytosis.
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Project supported by the National Basic Research Program of China (No 2004CB518802) and the Science and Technology Commission of Shanghai Municipality (No 054119568).
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Jiang, H., Zhang, J., Shi, Bz. et al. Application of EGFP-EGF fusions to explore mechanism of endocytosis of epidermal growth factor. Acta Pharmacol Sin 28, 111–117 (2007). https://doi.org/10.1111/j.1745-7254.2007.00481.x
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DOI: https://doi.org/10.1111/j.1745-7254.2007.00481.x
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