A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes
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- 作者：Tao Ke (1) (2)
Su Liang (3)
Jin Huang (3)
Han Mao (2)
Jibao Chen (1)
Caihua Dong (2)
Junyan Huang (2)
Shengyi Liu (2)
Jianxiong Kang (4)
Dongqi Liu (4)
Xiangdong Ma (3) - 关键词：antimicrobial peptide ; high throughput ; Npro ; prokaryotic expression
- 刊名：BMC Biotechnology
- 出版年：2012
- 出版时间：December 2012
- 年：2012
- 卷：12
- 期：1
- 全文大小：488KB
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Su Liang (3)
Jin Huang (3)
Han Mao (2)
Jibao Chen (1)
Caihua Dong (2)
Junyan Huang (2)
Shengyi Liu (2)
Jianxiong Kang (4)
Dongqi Liu (4)
Xiangdong Ma (3)
1. Department of Life Science and Technology, Nanyang Normal University, Wolong Road, Nanyang, 473061, China
2. Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, No.2 Xudong Second Road, Wuhan, 430062, China
3. Hubei Key Laboratory of Industrial Biotechnology, College of Life Science, Hubei University, Wuhan, 430062, China
4. School of Environmental Science and Engineering, Huazhong University of Science and Technology, Wuhan, 430074, China - ISSN：1472-6750
文摘
Background To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity. Results Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia. Conclusions The method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.