Construction of an energized fusion protein LDP-Hr-AE targeting human epidermal growth factor receptor 2 and its antitumor activity
- Author:
Xiao-Fang GUO
1
Author Information
1. Xinxiang Medical University
- Publication Type:Journal Article
- Keywords:
Antitumor;
Cell apoptosis;
Fusion protein;
Human epidermal growth factor receptor 2;
Lidamycin
- From:
Chinese Pharmaceutical Journal
2012;47(12):941-947
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE: To construct a novel fusion protein consisting of oligopeptides specific for human epidermal growth factor receptor 2(HER2) and lidamycin(LDM), and investigate its antitumor activity. METHODS: Coding sequences of oligopeptides from complementarity determining region 3(CDR3) of anti-HER2 antibody C6.5 heavy chain was fused to apoprotein of lidamycin to obtain the fusion gene ldp-Hr. Fusion protein LDP-Hr was expressed in E. coli and purified by affinity chromatography. The purity of LDP-Hr was analyzed by high HPLC. Immunofluorescence assay and flow cytometry-based binding assay were used to investigate the binding activity of LDP-Hr to HER2 overexpressed cancer cells. The energized fusion protein LDP-Hr-AE was prepared by integrating the active enediyne chromophore (AE) of lidamycin into the LDP-Hr protein. MTT assay was used to measure the in vitro cytotoxicity of LDP-Hr-AE and Annexin V-FITC/PI staining assay was used to analyze its apoptosis-inducing efficacy. RESULTS: Fusion protein LDP-Hr was constructed correctly and expressed in E. coli in a secretory manner. The production of LDP-Hr was 40 mg per liter fermentation broth, and the purity of fusion protein was 97.4% as analyzed by HPLC. LDP-Hr showed strong binding activity to cancer cells highly expressing HER2, such as SK-BR-3 and SK-OV-3 cells. The energized fusion protein LDP-Hr-AE exhibited more potent cytotoxicity to SK-BR-3 and SK-OV-3 cells than LDM as measured by MTT assay. The results from Annexin V-FITC/PI staining assay also revealed that LDP-Hr-AE significantly induced cell apoptosis even at very low concentrations. CONCLUSION: The novel energized fusion protein LDP-Hr-AE bounds to HER2 specifically, and shows potent cytotoxicity and apoptosis-inducing activity to cancer cells, which suggests that it would be a promising candidate for targeted cancer therapy.
