Rapid and efficient production of cecropin A antibacterial peptide in Escherichia coli by fusion with a self-aggregating protein

Meng Wang,Kaiwen Zheng,Jinglian Lin,Minhua Huang,Yi Ma,Xiaochun Luo,Shan Li,Jufang Wang

doi:10.1186/s12896-018-0473-7

Meng Wang, Kaiwen Zheng + Show 6 more

Open Access

https://doi.org/10.1186/s12896-018-0473-7

Copy DOI

Abstract

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.

Highlights

Cecropin 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
Design and construction of three different CeA fusions Because Antimicrobial peptides (AMPs) are toxic to host cells and are readily degraded by proteases [32], various proteins such as small ubiquitin-related modifier (SUMO) [33, 34], glutathione S-transferase (GST) [35], and TRX [31, 36] were selected as fusion partners for the efficient expression of AMPs
Based on the results of this study, ELK16 was selected and attached to the C-terminus of Mxe GyrA intein (CeA–Mxe–ELK16) in the SA-ELK16 system to induce and increase the formation of active fusion protein aggregates in E. coli. These aggregates were separated by simple centrifugation and high-purity CeA peptide was successfully released into soluble fractions after dithiothreitol (DTT)-induced Mxe GyrA intein cleavage and acetic acid treatment (Fig. 1b)

Summary

Introduction

Cecropin 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. Wang et al BMC Biotechnology (2018) 18:62 from insects [12,13,14] It contains a strongly cationic region at its N-terminus and a large hydrophobic tail at its C-terminus, which allow its interaction with the microbial membrane, and induces cell lysis by pore formation [1]. It has a wide spectrum of antimicrobial activities against Gram-negative bacteria, Gram-positive bacteria, and fungal phytopathogens [15]. The CeA peptide has become a potentially useful antimicrobial substance in biochemical and pharmaceutical research [17]

Methods

Results

Discussion

Conclusion