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| Fusion Protein Identification as Polymer and Its Analysis of Structure |
| NI Bei-bei, FAN Zhen-zhen, CHEN Hong, HUANG Bing-ren |
| Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, National Laboratory of Medical Molecular Biology, Beijing 100005, China |
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Abstract Objective: To confirm the polymer structure of the recombinant fusion protein EGF-E4orf4 and its EGFR binding activity. Methods: The gel filtration chromatogram,SDS-PAGE,Western blotting, light scattering, circular dichroism and receptor binding test were used to determine the characters of the protein EGF-E4orf4. Results: It was showed that the fusion protein EGF-E4orf4 formed a polymer. The static light scattering and dynamic light scattering determined that the molecular weight of EGF-E4orf4 is 1.656×107Da and the molecular radius is about 67.90nm, which indicated that EGF-E4orf4 polymerized with 820 monomers. The secondary structure of EGF-E4orf4 was riched in β sheet. Flow cytometry analysis proved that EGF-E4orf4 can combine with the cell surface EGFR. Conclusion: The fusion protein EGF-E4orf4 exists as a polymer and it could bind to EGFR. Therefore, as a potential novel targeted nticancer drug, EGF-E4orf4 has ossibility of clinical application.
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Received: 09 April 2012
Published: 25 July 2012
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[1] Ciardiello F, Tortora G A. Novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res, 2001, 7(10):2958-2970.
[2] Baselga J. Why the epidermal growth factor receptor? The rationale for cancer therapy. Oncologist, 2002, 7(Suppl 4):2-8.
[3] 廖刚. 靶向表皮生长因子受体的肿瘤生物治疗方法研究进展.中国肿瘤生物治疗杂志, 2009, 16(1): 97-100. Liao G. Epidermal growth factor receptor-targeted tumor biotherapies: an advance. Chin J Cancer Biother, 2009, 16(1): 97-100.
[4] Haigler H T, McKanna J A, Cohen S. Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J Cell Biol, 1979, 81(2):382-395.
[5] Longva K E, Blystad F D, Stang E, et al. Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies. J Cell Biol, 2002, 156(5): 843-854.
[6] Wang D M, Zhou Y, Xie H J, et al. Cytotoxicity of a recombinat fusion protein of adenovirus early region 4 oper reading frame 4 (E4orf4) and human epidermal growth factor of p53-deficient tumor cells. Anti-Cancer drugs, 2006, 17(5): 527-537.
[7] 卜鹏莉,王东梅,陈虹,等. 靶向性抗肿瘤融合蛋白EGF-E4orf4的表达及其细胞毒性的初步分析. 癌症, 2005, 24(1): 33-39. Bu P L, Wang D M, Chen H, et al. Expression of Epidermal Growth Factor-Adenovirus E4orf4 Fusion Protein in Tumor Cells and Its Cytotoxicity. Chinese Journal of Cancer, 2005, 24(1): 33-39.
[8] Zhou Y, Chen H, Ma X L, et al. Fusion protein of adenovirus E4orf4 and human epidermal growth factor inhibits tumor cell growth. Int J Cancer, 2009, 125(5): 1186-1192.
[9] Nakamura T, Takasugi H, Aizawa T, et al. Peptide mimics of epidermal growth factor (EGF) with antagonistic activity. J Biotechnol, 2005, 116(3):211-219.
[10] Ogiso H, Ishitani R, Nureki O, et al. Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell, 2002, 110(6):775-787.
[11] Danen-Van Oorschot A A, Fischer D F, Grimbergen J M, et al. Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells. Proc Natl Acad Sci U S A, 1997,94(11):5843-5847.
[12] Leliveld S R, Zhang Y H, Rohn J L, et al. Apoptin induces tumor-specific apoptosis as a globular multimer. J Biol Chem, 2003,278(11):9042-9051.
[13] 黄秉仁,蔡良婉,王欣,等. 甲基营养型酵母系统表达的重组人表皮生长因子的纯化及其性质. 中国医学科学院学报,2001, 23(2):106-110. Huang B R, Cai L W, Wang X, et al. Purification of recombinant hEGF expressed in yeast Pichia pastoris and the study on its characters. Acta Academiae Medicinae Sinicae, 2001, 23(2):106-110.
[14] 王存琳,陈虹,马晓骊,等. 靶向性抗肿瘤融合蛋白-表皮生长因子-腺病毒早期转录区4第4编码蛋白在细胞内的转运及对胞外信号调节激酶磷酸化的影响. 中国医学科学院学报, 2009, 31(6):674-678. Wang C L, Chen H, Ma X L, et al. Trafficking pathway and affectivity on extracellular signal-regulated kinase phosphorylation of anti-tumor fusion protein of epidermal growth factor – adenovirus early region 4 open reading frame 4 protein. Acta Academiae Medicinae Sinicae, 2009, 31(6):674-678. |
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