背景:尽管大肠杆菌已被广泛用于外源蛋白的表达,在这个系统中的分泌表达仍然是一个很大的障碍。作为最重要的分泌途径之一,大肠杆菌的溶血素A(HlyA)系统可以将底物直接从细胞质运输到细胞外培养基,而不会形成任何周质中间体,使其成为开发外源蛋白分泌生产平台的理想候选者。
结果:在这项工作中,我们开发了一个新颖的生产平台,Thhly,基于HlyA分泌系统,并探索了其在标签肽和抗菌肽的高效制备和快速检测中的应用。在这个新的平台中,HlyA的信号序列融合到目标肽的C末端,具有烟草蚀刻病毒(TEV)蛋白酶切割位点和它们之间的6*His标签。五种标签肽在大肠杆菌BL21(DE3)中显示出良好的分泌特性,其中T7标签和S标签通过两轮纯化步骤和TEV裂解获得,并保持其固有的免疫原性。此外,天蚕素A和蜂毒素,两种不同类型的广泛探索的抗微生物肽,同样产生并证实具有抗微生物/抗肿瘤生物活性。在融合蛋白表达期间未观察到显著的细菌生长抑制,表明融合形式不仅介导了分泌,而且降低了抗微生物肽(AMPs)对宿主细菌的毒性。据我们所知,这是首次报道在大肠杆菌中实现这两种AMP的分泌表达,具有相当大的制造和工业化潜力。
结论:结果表明,基于HlyA的新型大肠杆菌生产平台允许肽的有效分泌生产和纯化,从而为肽药物或试剂的工业化生产提出了有希望的策略。
BACKGROUND: Although Escherichia coli has been widely used for the expression of exogenous proteins, the secretory expression in this system is still a big obstacle. As one of the most important secretion pathways, hemolysin A (HlyA) system of E. coli can transport substrates directly from the cytoplasm to extracellular medium without the formation of any periplasmic intermediate, making it an ideal candidate for the development of the secretory production platform for exogenous proteins.
RESULTS: In this work, we developed a novel production platform, THHly, based on the HlyA secretion system, and explored its applications in the efficient preparation and quick detection of tag peptides and anti-microbial peptides. In this novel platform the signal sequence of HlyA is fused to the C-terminal of target peptide, with Tobacco Etch Virus (TEV) protease cleavage site and 6*His tag between them. Five tag peptides displayed good secretory properties in E. coli BL21 (DE3), among which T7 tag and S tag were obtained by two rounds of purification steps and TEV cleavage, and maintained their intrinsic immunogenicity. Furthermore, Cecropin A and Melittin, two different types of widely explored anti-microbial peptides, were produced likewise and verified to possess anti-microbial/anti-tumor bioactivities. No significant bacterial growth inhibition was observed during the fusion protein expression, indicating that the fusion form not only mediated the secretion but also decreased the toxicity of anti-microbial peptides (AMPs) to the host bacteria. To the best of our knowledge, this is the first report to achieve the secretory expression of these two AMPs in E. coli with considerable potential for manufacturing and industrialization purposes.
CONCLUSIONS: The results demonstrate that the HlyA based novel production platform of E. coli allowed the efficient secretory production and purification of peptides, thus suggesting a promising strategy for the industrialized production of peptide pharmaceuticals or reagents.