A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes

文献类型： 外文期刊

第一作者： Ke, Tao

作者： Ke, Tao;Chen, Jibao;Liang, Su;Huang, Jin;Ma, Xiangdong;Kang, Jianxiong;Liu, Dongqi;Ke, Tao;Mao, Han;Dong, Caihua;Huang, Junyan;Liu, Shengyi

作者机构：

关键词： antimicrobial peptide;high throughput;Npro;prokaryotic expression

期刊名称：BMC BIOTECHNOLOGY （ 影响因子：2.563； 五年影响因子：3.292 ）

ISSN： 1472-6750

年卷期： 2012 年 12 卷

页码：

收录情况： SCI

摘要： 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.

分类号：

  • 相关文献

[1]Multi-mycotoxin analysis of animal feed and animal-derived food using LC-MS/MS system with timed and highly selective reaction monitoring. Zhao, Zhiyong,Yang, Lingchen,Deng, Yifeng,Song, Suquan,Zhou, Zhenlei,Hou, Jiafa,Zhao, Zhiyong,Liu, Na,Wang, Jianhua,Lin, Shanhai,Wu, Aibo.

[2]Transgenic rice expressing a novel phytase-lactoferricin fusion gene to improve phosphorus availability and antibacterial activity. Deng Li-hua,Weng Lu-shui,Deng Xiang-yang,Xiao Guo-ying,Wang Zuo-ping,Fu Xi-qin,Xin Ye-yun. 2017

[3]The in vitro, in vivo antifungal activity and the action mode of Jelleine-I against Candida species. Jia, Fengjing,Wang, Jiayi,Peng, Jinxiu,Zhao, Ping,Wang, Kairong,Yan, Wenjin,Wang, Rui,Kong, Ziqing. 2018

[4]Identification of a cysteine-rich antimicrobial peptide from salivary glands of the tick Rhipicephalus haemaphysaloides. Zhang, Houshuang,Zhang, Wenjie,Wang, Xinzhi,Zhou, Yongzhi,Wang, Na,Zhou, Jinlin. 2011

[5]Design and pharmacodynamics of recombinant NZ2114 histidine mutants with improved activity against methicillin-resistant Staphylococcus aureus. Chen, Huixian,Feng, Xingjun,Chen, Huixian,Mao, Ruoyu,Teng, Da,Wang, Xiumin,Hao, Ya,Wang, Jianhua,Chen, Huixian,Mao, Ruoyu,Teng, Da,Wang, Xiumin,Hao, Ya,Wang, Jianhua. 2017

[6]Purification of antimicrobial peptide from Antarctic Krill (Euphausia superba) and its function mechanism. Zhao Ling,Yin Bangzhong,Liu Qi,Cao Rong. 2013

[7]Functional analysis of a novel cysteine-rich antimicrobial peptide from the salivary glands of the tick Rhipicephalus haemaphysaloides. Zhang, Houshuang,Yang, Siqi,Gong, Haiyan,Cao, Jie,Zhou, Yongzhi,Zhou, Jinlin,Zhou, Jinlin,Yang, Siqi.

[8]Purification and characterization of a novel antimicrobial peptide from Brevibacillus laterosporus strain A60. Guo, Lihua,Zeng, Hongmei,Yang, Xiufen,Yuan, Jingjing,Shi, Huaixing,Xiong, Yehui,Chen, Mingjia,Han, Lei,Qiu, Dewen.

[9]Design and recombination expression of a novel plectasin-derived peptide MP1106 and its properties against Staphylococcus aureus. Cao, Xintao,Zhang, Yong,Mao, Ruoyu,Teng, Da,Wang, Xiumin,Wang, Jianhua,Cao, Xintao,Zhang, Yong,Mao, Ruoyu,Teng, Da,Wang, Xiumin,Wang, Jianhua.

[10]Higher efficiency soluble prokaryotic expression, purification, and structural analysis of antimicrobial peptide G13. Che, Yuanyuan,Lu, Yinghu,Zha, Xiangdong,Ma, Lijuan,Xu, Xuejiao,Huang, Huoqing,Yang, Peilong.

[11]Multimerization and fusion expression of bovine lactoferricin derivative LfcinB15-W4,10 in Escherichia coli. Tian, Zi-gang,Da Teng,Yang, Ya-lin,Luo, Jin,Feng, Xing-jun,Fan, Ying,Zhang, Fan,Wang, Jian-hua.

[12]Two novel antimicrobial peptides purified from the symbiotic bacteria Xenorhabdus budapestensis NMC-10. Xiao, Yao,Meng, Fanlu,Qiu, Dewen,Yang, Xiufen.

[13]TC38, a teleost TFPI-2 peptide that kills bacteria via penetration of the cell membrane and interaction with nucleic acids. Zhang, Min,Yue, Bin,Wang, Guang-hua,Liu, Yong,Zhou, Shun,Cheng, Shun-feng,Li, Ning-qiu,Zhang, Ai-hua.

[14]D-amino acid substitution enhances the stability of antimicrobial peptide polybia-CP. Jia, Fengjing,Wang, Jiayi,Peng, Jinxiu,Zhao, Ping,Wang, Kairong,Yan, Wenjin,Wang, Rui,Kong, Ziqing. 2017

[15]Modification and characterization of a new recombinant marine antimicrobial peptide N2. Yang, Na,Feng, Xingjun,Wang, Xiumin,Teng, Da,Mao, Ruoyu,Hao, Ya,Zong, Lifen,Wang, Jianhua,Yang, Na,Wang, Xiumin,Teng, Da,Mao, Ruoyu,Hao, Ya,Zong, Lifen,Wang, Jianhua. 2016

[16]Improved antibacterial activity of a marine peptide-N2 against intracellular Salmonella typhimurium by conjugating with cell-penetrating peptides-bLFcin(6)/Tat(11). Li, Zhanzhan,Wang, Xiao,Teng, Da,Mao, Ruoyu,Hao, Ya,Yang, Na,Chen, Huixian,Wang, Xiumin,Wang, Jianhua,Li, Zhanzhan,Wang, Xiao,Teng, Da,Mao, Ruoyu,Hao, Ya,Yang, Na,Chen, Huixian,Wang, Xiumin,Wang, Jianhua. 2018

[17]High-level secretory expression of metchnikowin in Escherichia coli. Wu, Di,Lu, Yinghu,Ma, Lijuan,Che, Yuanyuan,Zha, Xiangdong,Yao, Bin,Huang, Huoqing,Yang, Peilong. 2013

[18]Construction of Salmonella Pullorum ghost by co-expression of lysis gene E and the antimicrobial peptide SMAP29 and evaluation of its immune efficacy in specific-pathogen-free chicks. Tian Qiu-feng,Zhou Wei,Si Wei,Yi Fei,Hua Xin,Chen Li-ping,Liu Si-guo,Yu Shen-ye,Tian Qiu-feng,Zhou Wei,Yi Fei,Yue Min. 2018

[19]GAP-initiated constitutive expression of a novel plectasin-derived peptide MP1106 by Pichia pastoris and its activity against Streptococcus suis. Jiao, Jian,Feng, Xingjun,Jiao, Jian,Mao, Ruoyu,Wang, Xiumin,Zhang, Yong,Teng, Da,Wang, Jianhua,Jiao, Jian,Mao, Ruoyu,Wang, Xiumin,Zhang, Yong,Teng, Da,Wang, Jianhua. 2015

[20]Recombinant production of the antimicrobial peptide NZ17074 in Pichia pastoris using SUMO3 as a fusion partner. Wang, X. J.,Wang, X. M.,Teng, D.,Zhang, Y.,Mao, R. Y.,Wang, J. H.,Wang, X. J.,Wang, X. M.,Teng, D.,Zhang, Y.,Mao, R. Y.,Wang, J. H..

作者其他论文 更多>>

微信小程序