As antibiotic-resistant bacterial pathogens become an ever-increasing concern, antimicrobial peptides (AMPs) have grown increasingly attractive as alternatives. Potentially, plants could be used as cost-effective AMP bioreactors; however, reported heterologous AMP expression is much lower in plants than in E. coli expression systems and often results in plant cytotoxicity, even for AMPs fused to carrier proteins. This suggests that there may be a physical characteristic of the previously described heterologous AMPs which impedes efficient expression in plants. Using a meta-analysis of protein databases, this study has determined that native plant AMPs were significantly less cationic than AMPs native to other taxa. To apply this finding to plant expression, the transient expression of 10 different heterologous AMPs, ranging in charge from +7 to -5, was tested in the tobacco, Nicotiana benthamiana. Elastin-like polypeptide (ELP) was used as the carrier protein for AMP expression. ELP fusion allowed for a simple, cost-effective temperature shift purification. Using this system, all five anionic AMPs expressed well, with two at unusually high levels (375 and 563 µg/gfw). Furthermore, antimicrobial activity against Staphylococcus epidermidis was an order of magnitude greater (average minimum inhibitory concentration MIC of 0.26 µM) than that typically seen for AMPs expressed in E. coli systems and was associated with the uncleaved fusion peptide. In summary, this study describes a means of expressing AMP fusions in plants in high yield, purified by a simple temperature-shift protocol, resulting in a fusion peptide with high antimicrobial activity and without the need for a peptide cleavage step.

中文翻译：

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将植物打造成具有成本效益的生物反应器，生产高活性抗菌肽


随着耐抗生素细菌病原体日益受到关注，抗菌肽 (AMP) 作为替代品变得越来越有吸引力。植物有可能用作具有成本效益的 AMP 生物反应器；然而，据报道植物中的异源 AMP 表达比大肠杆菌表达系统低得多，并且经常导致植物细胞毒性，即使对于与载体蛋白融合的 AMP 也是如此。这表明先前描述的异源 AMP 可能存在阻碍植物中有效表达的物理特征。通过对蛋白质数据库的荟萃分析，本研究确定天然植物 AMP 的阳离子性明显低于其他类群的天然 AMP。为了将这一发现应用于植物表达，在烟草 Benthamiana 中测试了 10 种不同的异源 AMP（电荷范围为 +7 至 -5）的瞬时表达。弹性蛋白样多肽（ELP）被用作AMP表达的载体蛋白。 ELP 融合可实现简单、经济高效的温度变换纯化。使用该系统，所有五种阴离子 AMP 均表达良好，其中两种的水平异常高（375 和 563 µg/gfw）。此外，针对表皮葡萄球菌的抗菌活性比大肠杆菌系统中表达的 AMP 的典型抗菌活性高一个数量级（平均最小抑制浓度 MIC 为 0.26 µM），并且与未切割的融合肽相关。总之，本研究描述了一种在植物中高产量表达 AMP 融合物的方法，通过简单的温度变换方案纯化，产生具有高抗菌活性的融合肽，并且不需要肽裂解步骤。