Production, purification, and characterization of the cecropin from Plutella xylostella, pxCECA1, using an intein-induced self-cleavable system in Escherichia coli

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• 作者：Hong Wang (1)
  Xiao-lin Meng (1) mengxiaolin8@hotmail.com
  Jin-ping Xu (1)
  Jian Wang (1)
  Hua Wang (2)
  Chun-wei Ma (2)
• 关键词：Plutella xylostella – Cecropin – Self ; cleavage – Antimicrobial activity
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2012
• 出版时间：May 2012
• 年：2012
• 卷：94
• 期：4
• 页码：1031-1039
• 全文大小：372.0 KB
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• 作者单位：1. State Key Laboratory of Virology, College of Life Science, Wuhan University, Wuhan, 430072 People’s Republic of China2. Zhongnan Hospital of Wuhan University, Wuhan, 430072 People’s Republic of China
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

Antimicrobial peptides (AMPs) are widely expressed and play an important role in innate immune defense against infectious agents such as bacteria, viruses, fungi, and parasites. Cecropins are a family of AMPs synthesized in the fat body of insects that have proven effective at killing specific pathogens. In order to fulfill their clinical potential as antimicrobial drugs, a simple, cost-effective method to express AMPs is sorely needed. In this study, we expressed and characterized the cecropin from Plutella xylostella (pxCECA1) using an intein-dependent expression system in Escherichia coli. We cloned the pxCECA1 gene from larva by RT-PCR and fused the encoding sequence of mature pxCECA1 with an intein gene and a chitin-binding domain gene (CBD) in pTWIN1 plasmid. The fusion protein CBD–intein–pxCECA1 was expressed in E. coli BL21 (DE3) and separated by flowing cell extracts through a chitin column. Subsequently, self-cleavage of the intein at its C-terminus was induced in a temperature- and pH-dependent manner, resulting in the release of mature pxCECA1. The optimal conditions for self-cleavage were determined to be pH 6.0 for 48 h at 4°C, under which 12.3 mg of recombinant pxCECA1 could be recovered from 1 l of E. coli culture. The purified pxCECA1 displayed antimicrobial activity against a broad variety of gram-positive and gram-negative bacteria. This preparation was especially effective against Staphylococcus aureus, including methicillin-resistant strains. Catalase release assays demonstrated that pxCECA1 acts as a microbicidal agent. These results show for the first time that the IMPACT-TWIN expression system is an efficient, cost-effective way to produce fully functional AMPs and that the AMP pxCECA1 is a novel microbicidal agent with promising therapeutic applications.