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

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.