School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
BMC Biotechnol. 2018 Oct 5;18(1):62. doi: 10.1186/s12896-018-0473-7.
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.
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.
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.
抗菌肽 Cecropin A (CeA) 是一种天然阳离子抗菌肽，对革兰氏阳性和革兰氏阴性菌具有广谱的抗菌活性，因此成为替代抗生素的有吸引力的候选物质。然而，CeA 的重组和化学合成都需要低产量和繁琐、昂贵的工艺，这严重阻碍了 CeA 的开发和应用。在这里，我们利用一种短的 β 结构自聚集蛋白 ELK16 作为 CeA 的融合伴侣，利用简单廉价的工艺高效生产高纯度的 CeA 抗菌肽。
本研究考察了三种不同的 CeA 肽生产方法：亲和标签（His 标签）融合蛋白表达系统（AT-HIS 系统）、无细胞蛋白表达系统（CF 系统）和自组装肽（ELK16）融合蛋白表达系统（SA-ELK16 系统）。在 AT-HIS 和 CF 系统中，通过一种或多种亲和层析纯化步骤，CeA 肽的纯度分别达到 92.1%和 90.4%。这些步骤繁琐且成本高昂，CeA 的产率分别仅为 0.41 和 0.93μg/mg 湿细胞重量。令人惊讶的是，在 SA-ELK16 系统中，通过简单廉价的包括离心和醋酸处理等步骤，获得了约 6.2μg/mg 湿细胞重量的高纯度（约 99.8%）CeA 肽。抗菌试验表明，本研究中制备的高纯度 CeA 具有与合成 CeA 肽相同的抗菌活性。
本研究设计了一种合适的抗菌肽 CeA 表达系统（SA-ELK16 系统），并与另外两种蛋白表达系统进行了比较。通过 SA-ELK16 系统获得了高产率的高纯度 CeA 肽，大大降低了下游处理所需的成本和时间。该系统可为 CeA 抗菌肽的实验室规模生产提供平台。
