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A novel secretion and online-cleavage strategy for production of cecropin A in Escherichia coli.

ABSTRACT: Antimicrobial peptides, promising antibiotic candidates, are attracting increasing research attention. Current methods for production of antimicrobial peptides are chemical synthesis, intracellular fusion expression, or direct separation and purification from natural sources. However, all these methods are costly, operation-complicated and low efficiency. Here, we report a new strategy for extracellular secretion and online-cleavage of antimicrobial peptides on the surface of Escherichia coli, which is cost-effective, simple and does not require complex procedures like cell disruption and protein purification. Analysis by transmission electron microscopy and semi-denaturing detergent agarose gel electrophoresis indicated that fusion proteins contain cecropin A peptides can successfully be secreted and form extracellular amyloid aggregates at the surface of Escherichia coli on the basis of E. coli curli secretion system and amyloid characteristics of sup35NM. These amyloid aggregates can be easily collected by simple centrifugation and high-purity cecropin A peptide with the same antimicrobial activity as commercial peptide by chemical synthesis was released by efficient self-cleavage of Mxe GyrA intein. Here, we established a novel expression strategy for the production of antimicrobial peptides, which dramatically reduces the cost and simplifies purification procedures and gives new insights into producing antimicrobial and other commercially-viable peptides.

SUBMITTER: Wang M

PROVIDER: S-EPMC5544755 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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A novel secretion and online-cleavage strategy for production of cecropin A in Escherichia coli.

Wang Meng M Huang Minhua M Zhang Junjie J Ma Yi Y Li Shan S Wang Jufang J

Scientific reports 20170804 1

Antimicrobial peptides, promising antibiotic candidates, are attracting increasing research attention. Current methods for production of antimicrobial peptides are chemical synthesis, intracellular fusion expression, or direct separation and purification from natural sources. However, all these methods are costly, operation-complicated and low efficiency. Here, we report a new strategy for extracellular secretion and online-cleavage of antimicrobial peptides on the surface of Escherichia coli, w ...[more]

PMID: 28779147

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Project description:BackgroundCecropin A (CeA), a natural cationic antimicrobial peptide, exerts potent antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria, making it an attractive candidate substitute for antimicrobials. However, the low production rate and cumbersome, expensive processes required for both its recombinant and chemical synthesis have seriously hindered the exploitation and application of CeA. Here, we utilized a short β-structured self-aggregating protein, ELK16, as a fusion partner of CeA, which allowed the efficient production of high-purity CeA antibacterial peptide with a simple inexpensive process.ResultsIn this study, three different approaches to the production of CeA peptide were investigated: an affinity tag (His-tag)-fused protein expression system (AT-HIS system), a cell-free protein expression system (CF system), and a self-assembling peptide (ELK16)-fused protein expression system (SA-ELK16 system). In the AT-HIS and CF systems, the CeA peptide was obtained with purities of 92.1% and 90.4%, respectively, using one or more affinity-chromatographic purification steps. The procedures were tedious and costly, with CeA yields of only 0.41 and 0.93 μg/mg wet cell weight, respectively. Surprisingly, in the SA-ELK16 system, about 6.2 μg/mg wet cell weight of high-purity (approximately 99.8%) CeA peptide was obtained with a simple low-cost process including steps such as centrifugation and acetic acid treatment. An antimicrobial test showed that the high-purity CeA produced in this study had the same antimicrobial activity as synthetic CeA peptide.ConclusionsIn this study, we designed a suitable expression system (SA-ELK16 system) for the production of the antibacterial peptide CeA and compared it with two other protein expression systems. A high yield of high-purity CeA peptide was obtained with the SA-ELK16 system, which greatly reduced the cost and time required for downstream processing. This system may provide a platform for the laboratory scale production of the CeA antibacterial peptide.

Dataset Information

A novel secretion and online-cleavage strategy for production of cecropin A in Escherichia coli.

Publications

A novel secretion and online-cleavage strategy for production of cecropin A in Escherichia coli.

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