[Construction of energized fusion protein consisting of epidermal growth factor receptor oligopeptide ligand and lidamycin and its antitumor activity].
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.


CONCLUSIONS
The energized fusion protein Ec-LDP-AE binds to EGFR specifically and kills cancer cells efficiently.
Guo
Xiao-Fang
XF
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Perking Union Medical College, Beijing, 100050, P.R. China.
Zhong
Gen-Shen
GS
Miao
Qing-Fang
QF
Zhen
Yong-Su
YS
D000617:Aminoglycosides / Q000494:pharmacology*; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D053281:Enediynes / Q000494:pharmacology*; D066246:ErbB Receptors / Q000235:genetics / Q000494:pharmacology*; D005260:Female; D006801:Humans; D008024:Ligands; D051379:Mice; D041681:NIH 3T3 Cells; D009842:Oligopeptides / Q000235:genetics / Q000494:pharmacology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology
D000617:Aminoglycosides; D000903:Antibiotics, Antineoplastic; D053281:Enediynes; D008024:Ligands; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C058024:C 1027; D066246:ErbB Receptors