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

ABSTRACT: 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.

SUBMITTER: Ke T

PROVIDER: S-EPMC3350388 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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

Ke Tao T Liang Su S Huang Jin J Mao Han H Chen Jibao J Dong Caihua C Huang Junyan J Liu Shengyi S Kang Jianxiong J Liu Dongqi D Ma Xiangdong X

BMC biotechnology 20120323

<h4>Background</h4>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, C ...[more]

PMID: 22439858

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Project description:BACKGROUND:Conventional plant DNA isolation methods are complex, time consuming and require technical expertise. These limitations were overcome using the DNA isolation kits which, however significantly add to the research costs. Hence the present study was aimed to develop a high throughput, rapid and inexpensive method of PCR ready DNA template preparation from plant materials. METHODS:Concentration of SDS in lysis buffer, amount of starting material, period and temperature for lysis were optimized for obtaining PCR ready templates from plant materials. The method was tested using RAPD and ITS specific primers for different plant species like rice, wheat, mustard, pea, soybean, pigeonpea, tomato, maize, march lilly, bougainvillea, Indian blanket flower, nerium, petunia, purple pirouette petunia, moses-in-the-cradle, golden cane palm, duranta, periwinkle, chrysanthemum and two xerophytes viz. Dipterygium glaucum and Crotaleria burhia. SSR markers RM18398 and RM26108 showed successful amplification in rice varieties Improved Pusa Basmati 1 and KS Dev 12. The effectiveness of the method was tested using fresh as well as 1 year old tissues. The storability of the lysate was also tested. RESULTS:In this report, we developed a novel method called rapid high throughput template preparation (rHTTP) method to prepare PCR ready DNA templates. Most striking feature of this technique is that it can be done anywhere where water can be boiled by any means. Using rHTTP method, PCR ready templates can be prepared in just 10 min. Robust and reproducible amplification for all the test plants were recorded with RAPD, plant ITS primers and SSR markers following this method. rHTTP methods works well for both fresh as well as old plant tissues. The lysates had a shelf life of 1 month when stored at 4 °C and 3 days when stored at room temperature. CONCLUSIONS:rHTTP method has several advantages over the other protocols like ease of execution, no requirement of tissue grinding/liquid nitrogen/hazardous chemicals and above all, equally effective for both fresh and old samples. Using this method, costs per prep comes down ~ 10-50 times as compared to most commercial kits. This method can be used for on-field experiments like molecular diagnostics, varietal identification etc.

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

Publications

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

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