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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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