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Construction of a membrane-lytic immunotoxin using melittin and epidermal growth factor

Published online by Cambridge University Press:  24 April 2009

Chen Mei-Ling ,
Niu Dong ,
Ruan Hui ,
Pan Bing-Qing ,
Wang Jin-Ling ,
Wu De ,
Zhang Jia-Jia ,
Chen Qi-He  and
He Guo-Qing
Chen Mei-Ling
Affiliation:
Bioengineering and Food Science College, Zhejiang University, Hangzhou 310029, China
Niu Dong
Affiliation:
Animal Science College, Zhejiang University, Hangzhou 310029, China
Ruan Hui*
Affiliation:
Bioengineering and Food Science College, Zhejiang University, Hangzhou 310029, China
Pan Bing-Qing
Affiliation:
Bioengineering and Food Science College, Zhejiang University, Hangzhou 310029, China
Wang Jin-Ling
Affiliation:
Bioengineering and Food Science College, Zhejiang University, Hangzhou 310029, China
Wu De
Affiliation:
Zhejiang Jinhua Provincial Hi-tech Park, Jinhua 321017, China
Zhang Jia-Jia
Affiliation:
Bioengineering and Food Science College, Zhejiang University, Hangzhou 310029, China
Chen Qi-He
Affiliation:
Bioengineering and Food Science College, Zhejiang University, Hangzhou 310029, China
He Guo-Qing
Affiliation:
Bioengineering and Food Science College, Zhejiang University, Hangzhou 310029, China
*
*Corresponding author. E-mail: [email protected]
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Abstract

Epidermal growth factor receptor (EGFR) is becoming a perfect target for killing carcinoma cells, especially because of its overexpression on the surface of these cells. Cationic antimicrobial peptides (CAP) have their own special mechanism of membrane-lytic cytotoxicity. In this study, a membrane-lytic immunotoxin (IT), chimeric protein MEGFMEL, was constructed to kill carcinoma cells with EGFR overexpression. This protein is composed of mouse (Mus musculus) epidermal growth factor (MEGF), as the target part, and melittin (MEL), as the cytotoxic part. Using Escherichia coli BL21 and pET30a as expression strain and vector, respectively, 63.45 μg/ml of MEGFMEL (68% purity) was obtained through low-temperature induction of expression and a thawing-freezing purification procedure (without cytolysis). In vitro activity measurement showed that this MEGFMEL significantly induced a lethal effect on A431 carcinoma cells overexpressing EGFR on the surface, with an LD50 value 52.6 μg/ml. The results suggest that the use of CAP as the toxin in the construction of unique membrane-lytic ITs aimed at EGFR is feasible.

Keywords

membrane-lytic immunotoxinmelittinepidermal growth factor receptor
Type
Research Papers
Information
Chinese Journal of Agricultural Biotechnology , Volume 6 , Issue 1 , April 2009 , pp. 35 - 39
Copyright
Copyright © China Agricultural University 2009

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References

Burgess, AW, Lloyd, CJ, Smith, S, et al. (1988) Murine epidermal growth factor: structure and function. Biochemistry 27: 4977–4985.CrossRefGoogle ScholarPubMed
Choe, S, Bennett, MJ, Fujii, G, et al. (1992) The crystal structure of diphtheria toxin. Nature 357: 216–222.CrossRefGoogle ScholarPubMed
Davies, DE and Chamberlin, SG (1996) Targeting the epidermal growth factor receptor for therapy of carcinomas. Biochemical Pharmacology 51: 1101–1110.CrossRefGoogle ScholarPubMed
Gullick, WJ (1991) Prevalence of aberrant expression of the epidermal growth factor receptor in human cancers. British Medical Bulletin 47(1): 87–98.CrossRefGoogle ScholarPubMed
Harris, RA, Eichholtz, TJ, Hiles, ID, Page, MJ and O'Hare, MJ (1999) New model of Erb-2 overexpression in human mammary luminal epithelial cells. International Journal of Cancer 80: 477–486.3.0.CO;2-W>CrossRefGoogle Scholar
He, GQ, Ruan, H, Niu, D, Chen, G, Yu, XP and Fu, Y (2002) Reverse transcription, amplification and cloning of melittin mature region of Apis cerana by Taq DNA polymerase. Journal of Agricultural Biotechnology 10(1): 85–97 (in Chinese).Google Scholar
Huang, HQ and Wang, YZ (2005) Gene cloning and Escherichia coli expression of murine lactoferrin. Journal of Agricultural Biotechnology 13(1): 52–55 (in Chinese).Google Scholar
Kersemaekers, AMF, Fleuren, GJ, Kenter, EG, et al. (1999) Oncogene alterations in carcinomas of the uterine cervix: overexpression of the epidermal growth factor receptor is associated with poor prognosis. Clinical Cancer Research 5: 577–586.Google ScholarPubMed
Kreitman, RJ (2001) Chimeric fusion proteins – pseudomonas exotoxin-based. Current Opinions in Investigation 2(9): 1282–1293.Google ScholarPubMed
Lambert, JM, Goldmacher, VS and Blattler, WA (1991) An immunotoxin prepared with blocked ricin: a natural plant toxin adapted for therapeutic use. Cancer Research 51: 6236–6242.Google ScholarPubMed
Li, GH, Zhang, Y and Li, N (2001) Analysis on the partial sequence of bovine lactoferrin gene by PCR–SSCP. Journal of Agricultural Biotechnology 9(2): 139–141 (in Chinese).Google Scholar
Modi, S and Seidman, AD (2002) An update on epidermal growth factor receptor inhibitors. Current Oncological Peptides 4(1): 47–55.Google ScholarPubMed
Pennell, CA and Erickson, HA (2002) Designing immunotoxins for cancer therapy. Immunological Research 25(2): 177–191.CrossRefGoogle ScholarPubMed
Ruan, H, Niu, D, Zhao, Y, He, GQ and Fu, Y (2005) Studies on the expression of mouse epidermal growth factor in E. coli. Journal of Agricultural Biotechnology 13(4): 468–471 (in Chinese).Google Scholar
Shi, WJ, Yang, ZN, Zhang, CX and Cheng, JA (2006) Expression of the melittin gene of Polistes hebraeus fused with enhanced green fluorescent protein gene in insect cells. Journal of Agricultural Biotechnology 11(1): 102–106 (in Chinese).Google Scholar
Wang, JZ and Fan, M (2000) The Protein Technique Manual. Beijing: Science Press, pp. 77–101 (in Chinese).Google Scholar
Wang, YZ, Han, FF and Huang, HQ (2004a) Cloning and sequence analysis of the lactoferrin cDNA from mice. Journal of Agricultural Biotechnology 12(2): 230–231 (in Chinese).Google Scholar
Wang, YZ, Lin, WX and Xu, ZR (2004b) Antibacterial peptide PR-39 gene expression in different growth stages of pig evaluated by semi-quantitative RT-PCR method. Journal of Agricultural Biotechnology 12(1): 52–55 (in Chinese).Google Scholar
Zhang, HH, Bi, YZ, Cao, YC, Ma, JY and Chen, XC (2005) Cloning and structure analysis of chicken beta defensin gene. Journal of Agricultural Biotechnology 13(4): 482–488 (in Chinese).Google Scholar
Zhao, YY, Liu, F, Zheng, H-Y, Lu, Q and Zheng, JG (2006) Expression and bioactivity of human lactoferrin gene in transgenic tomato. Journal of Agricultural Biotechnology 14(2): 299–300 (in Chinese).Google Scholar
Zhou, SW (1991) Experimental Biochemistry Techniques. Beijing: Beijing Agricultural University Press, pp. 41–44 (in Chinese).Google Scholar