Application of EGFP-EGF fusions to explore mechanism of endocytosis of epidermal growth factor
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.
Keywords:
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.