Guo Xiao-Fang, Zhong Gen-Shen, Miao Qing-Fang, Zhen Yong-Su
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Perking Union Medical College, Beijing, 100050, P.R. China.
Ai Zheng. 2009 Jun;28(6):561-8.
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.
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.
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.
The energized fusion protein Ec-LDP-AE binds to EGFR specifically and kills cancer cells efficiently.
表皮生长因子受体（EGFR）在多种肿瘤细胞上异常过度表达。力达霉素是一种具有高效抗肿瘤活性的烯二炔类抗生素。本研究旨在通过重组EGFR特异性寡肽配体和力达霉素构建一种新型融合蛋白，并研究其抗肿瘤疗效。
融合蛋白（Ec-LDP）在大肠杆菌中表达，通过亲和层析进行纯化。采用高效液相色谱（HPLC）分析Ec-LDP的纯度。运用酶联免疫吸附测定（ELISA）、流式细胞术（FCM）和免疫荧光分析来检测Ec-LDP与不同癌细胞系的结合活性。通过将力达霉素的活性烯二炔发色团（AE）整合到Ec-LDP蛋白中制备活性融合蛋白Ec-LDP-AE。采用MTT法测定Ec-LDP-AE的细胞毒性。
成功构建了Ec-LDP融合蛋白并在大肠杆菌中分泌表达，每升发酵液中Ec-LDP蛋白的产量为18 mg。Ec-LDP蛋白的纯度为95.3%。Ec-LDP蛋白对高表达EGFR的癌细胞系，如MCF-7和A431细胞具有较强的结合活性。然而，Ec-LDP对EGFR阴性的NIH 3T3细胞没有结合活性。活性融合蛋白Ec-LDP-AE对MCF-7和A431细胞显示出强大的细胞毒性，其半数抑制浓度（IC50）值分别为3.06×10⁻¹¹ mol/L和9.38×10⁻¹³ mol/L。
活性融合蛋白Ec-LDP-AE能特异性结合EGFR并有效杀伤癌细胞。
