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

作　　者：Hua JIANG Jie ZHANG Bi-zhi SHI Yu-hong XU Zong-hai LI Jian-ren GU 

机构地区：[1]State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Medical College of Fudan University [2]School of Pharmacy, Shanghai Jiaotong University, Shanghai 200032, China

出　　处：《Acta Pharmacologica Sinica》2007年第1期111-117,共7页中国药理学报（英文版）

基　　金：Project supported by the National Basic Research Program of China(No 2004CB518802);the Science and Technology Commission of Shanghai Municipality(No 054119568).

摘　　要：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 micro- scopy. 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.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 micro- scopy. 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.

关 键 词：receptor  epidermal growth factor ENDOCYTOSIS PURIFICATION expression 

分 类 号：R575[医药卫生—消化系统]