School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, China.
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, China.
Sci Rep. 2017 Aug 4;7(1):7368. doi: 10.1038/s41598-017-07411-5.
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.
抗菌肽是很有前途的抗生素候选物，越来越受到研究关注。目前抗菌肽的生产方法有化学合成、细胞内融合表达或直接从天然来源分离和纯化。然而，所有这些方法都成本高、操作复杂、效率低。在这里，我们报告了一种在大肠杆菌表面进行抗菌肽细胞外分泌和在线切割的新策略，该策略具有成本效益高、简单的特点，不需要细胞破碎和蛋白质纯化等复杂步骤。透射电子显微镜和半变性去污剂琼脂糖凝胶电泳分析表明，基于大肠杆菌卷曲菌分泌系统和 sup35NM 的淀粉样特性，融合蛋白包含抗菌肽 Cecropin A 可以成功地在大肠杆菌表面进行细胞外分泌，并形成细胞外淀粉样聚集物。这些淀粉样聚集物可以通过简单的离心轻松收集，通过 Mxe GyrA 内含子的有效自我切割释放出具有与商业合成肽相同抗菌活性的高纯度 Cecropin A 肽。在这里，我们建立了一种生产抗菌肽的新型表达策略，大大降低了成本，简化了纯化步骤，并为生产抗菌肽和其他有商业价值的肽提供了新的思路。
