BACKGROUND AND OBJECTIVE: Epidermal growth factor receptor (EGFR) is abnormally overexpressed on many kinds of tumor cells. Lidamycin is an enediyne antibiotic with highly potent antitumor activity. This study was to construct a novel fusion protein by recombining EGFR specific oligopeptide ligand and lidamycin, and investigate its antitumor efficacy. METHODS: The fusion protein (Ec-LDP) was expressed in E.coli and purified by affinity chromatography. The purity of Ec-LDP was analyzed by high performance liquid chromatography (HPLC). ELISA, flow cytometry (FCM) and immunofluorescence assay were used to analyze the binding activity of Ec-LDP to different cancer cell lines. The energized fusion protein Ec-LDP-AE was prepared by integrating the active enediyne chromophore (AE) of lidamycin into the Ec-LDP protein. The cytotoxicity of Ec-LDP-AE was measured by MTT assay. RESULTS: Ec-LDP fusion protein was successfully constructed and secretorily expressed in E.coli, and the production of Ec-LDP protein was 18 mg per liter fermentation broth. The purity of Ec-LDP protein was 95.3%. Ec-LDP protein had strong binding activity to cancer cell lines highly expressing EGFR, such as MCF-7 and A431 cells. However, Ec-LDP had no binding activity to EGFR negative NIH 3T3 cells. The energized fusion protein Ec-LDP-AE showed potent cytotoxicity to MCF-7 and A431 cells with the half maximal inhibitory concentration (IC50) values of 3.06 x 10(-11) mol/L and 9.38 x 10(-13) mol/L, respectively. CONCLUSION: The energized fusion protein Ec-LDP-AE binds to EGFR specifically and kills cancer cells efficiently.
BACKGROUND AND OBJECTIVE:Epidermal growth factor receptor (EGFR) is abnormally overexpressed on many kinds of tumor cells. Lidamycin is an enediyne antibiotic with highly potent antitumor activity. This study was to construct a novel fusion protein by recombining EGFR specific oligopeptide ligand and lidamycin, and investigate its antitumor efficacy. METHODS: The fusion protein (Ec-LDP) was expressed in E.coli and purified by affinity chromatography. The purity of Ec-LDP was analyzed by high performance liquid chromatography (HPLC). ELISA, flow cytometry (FCM) and immunofluorescence assay were used to analyze the binding activity of Ec-LDP to different cancer cell lines. The energized fusion protein Ec-LDP-AE was prepared by integrating the active enediyne chromophore (AE) of lidamycin into the Ec-LDP protein. The cytotoxicity of Ec-LDP-AE was measured by MTT assay. RESULTS: Ec-LDP fusion protein was successfully constructed and secretorily expressed in E.coli, and the production of Ec-LDP protein was 18 mg per liter fermentation broth. The purity of Ec-LDP protein was 95.3%. Ec-LDP protein had strong binding activity to cancer cell lines highly expressing EGFR, such as MCF-7 and A431 cells. However, Ec-LDP had no binding activity to EGFR negative NIH 3T3 cells. The energized fusion protein Ec-LDP-AE showed potent cytotoxicity to MCF-7 and A431 cells with the half maximal inhibitory concentration (IC50) values of 3.06 x 10(-11) mol/L and 9.38 x 10(-13) mol/L, respectively. CONCLUSION: The energized fusion protein Ec-LDP-AE binds to EGFR specifically and kills cancer cells efficiently.