Rational Design and Biotechnological Production of Novel AfpB-PAF26 Chimeric Antifungal Proteins
Rational Design and Biotechnological Production of Novel AfpB-PAF26 Chimeric Antifungal Proteins
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DOI：
10.3390/microorganisms6040106
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发表时间：
2018-12-01
期刊：
影响因子：
4.5
通讯作者：
Manzanares, Paloma
中科院分区：
文献类型：
--
作者：
Heredero, Marcos;Garrigues, Sandra;Manzanares, Paloma
抗菌肽已被认为是开发新的抗菌化合物的候选者，用于医学、农业和食品防腐。PAF26是一种合成的抗真菌六肽，通过组合方法获得，对丝状真菌具有有效的杀真菌活性。其他有趣的AMP是真菌来源的抗真菌蛋白（AFP），它们是富含半胱氨酸的碱性小蛋白，可以通过生物技术大量生产。一种有希望的AFP是在植物病原体指状青霉中鉴定的AfpB。在这项工作中，我们的目的是合理设计，生物技术生产和测试AFPB：：PAF26嵌合蛋白，以获得设计的AFP（dAfpBs）具有改善的性质。dAfpB6和dAfpB9嵌合体可以使用指状假单胞菌作为生物工厂和先前描述的基于产黄青霉的表达盒来产生，但仅dAfpB9可以被纯化和表征。蛋白dAfpB9显示出对丝状真菌的真菌抑制活性的微妙和真菌依赖性的差异相比，天然AFPB。值得注意的是，dAfpB9失去了PAF 26和AfpB的杀真菌活性，因此将该活性与抑真菌活性断开，并将杀真菌决定簇映射到AfpB的暴露环L3，其中定位有修饰。本研究为新型嵌合AFP的设计和开发提供了信息。
Antimicrobial peptides (AMPs) have been proposed as candidates to develop new antimicrobial compounds for medicine, agriculture, and food preservation. PAF26 is a synthetic antifungal hexapeptide obtained from combinatorial approaches with potent fungicidal activity against filamentous fungi. Other interesting AMPs are the antifungal proteins (AFPs) of fungal origin, which are basic cysteine-rich and small proteins that can be biotechnologically produced in high amounts. A promising AFP is the AfpB identified in the phytopathogen Penicillium digitatum. In this work, we aimed to rationally design, biotechnologically produce and test AfpB::PAF26 chimeric proteins to obtain designed AFPs (dAfpBs) with improved properties. The dAfpB6 and dAfpB9 chimeras could be produced using P. digitatum as biofactory and a previously described Penicillium chrysogenum-based expression cassette, but only dAfpB9 could be purified and characterized. Protein dAfpB9 showed subtle and fungus-dependent differences of fungistatic activity against filamentous fungi compared to native AfpB. Significantly, dAfpB9 lost the fungicidal activity of PAF26 and AfpB, thus disconnecting this activity from the fungistatic activity and mapping fungicidal determinants to the exposed loop L3 of AfpB, wherein modifications are located. This study provides information on the design and development of novel chimeric AFPs.