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

Abstract

Abstract Background 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. 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. Results In 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. Conclusions In 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.

Keywords

• Self-aggregating protein
• Cecropin A
• Antimicrobial peptide
• ELK16
• Escherichia coli