Rivera-de-Torre Esperanza, Rimbault Charlotte, Jenkins Timothy P, Sørensen Christoffer V, Damsbo Anna, Saez Natalie J, Duhoo Yoan, Hackney Celeste Menuet, Ellgaard Lars, Laustsen Andreas H
Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.
Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia.
Front Bioeng Biotechnol. 2022 Jan 20;9:811905. doi: 10.3389/fbioe.2021.811905. eCollection 2021.
Animal venoms are complex mixtures containing peptides and proteins known as toxins, which are responsible for the deleterious effect of envenomations. Across the animal Kingdom, toxin diversity is enormous, and the ability to understand the biochemical mechanisms governing toxicity is not only relevant for the development of better envenomation therapies, but also for exploiting toxin bioactivities for therapeutic or biotechnological purposes. Most of toxinology research has relied on obtaining the toxins from crude venoms; however, some toxins are difficult to obtain because the venomous animal is endangered, does not thrive in captivity, produces only a small amount of venom, is difficult to milk, or only produces low amounts of the toxin of interest. Heterologous expression of toxins enables the production of sufficient amounts to unlock the biotechnological potential of these bioactive proteins. Moreover, heterologous expression ensures homogeneity, avoids cross-contamination with other venom components, and circumvents the use of crude venom. Heterologous expression is also not only restricted to natural toxins, but allows for the design of toxins with special properties or can take advantage of the increasing amount of transcriptomics and genomics data, enabling the expression of dormant toxin genes. The main challenge when producing toxins is obtaining properly folded proteins with a correct disulfide pattern that ensures the activity of the toxin of interest. This review presents the strategies that can be used to express toxins in bacteria, yeast, insect cells, or mammalian cells, as well as synthetic approaches that do not involve cells, such as cell-free biosynthesis and peptide synthesis. This is accompanied by an overview of the main advantages and drawbacks of these different systems for producing toxins, as well as a discussion of the biosafety considerations that need to be made when working with highly bioactive proteins.
动物毒液是一种复杂的混合物，包含被称为毒素的肽和蛋白质，这些毒素是造成中毒有害影响的原因。在整个动物界，毒素的多样性极为丰富，理解控制毒性的生化机制不仅与开发更好的中毒治疗方法相关，而且对于利用毒素的生物活性用于治疗或生物技术目的也很重要。大多数毒素学研究依赖于从粗毒液中获取毒素；然而，一些毒素难以获得，原因在于有毒动物濒临灭绝、在圈养环境中无法茁壮成长、仅产生少量毒液、难以采集毒液，或者仅产生少量感兴趣的毒素。毒素的异源表达能够产生足够量的毒素，从而挖掘这些生物活性蛋白的生物技术潜力。此外，异源表达确保了同质性，避免了与其他毒液成分的交叉污染，并规避了粗毒液的使用。异源表达不仅限于天然毒素，还允许设计具有特殊性质的毒素，或者可以利用越来越多的转录组学和基因组学数据，实现休眠毒素基因的表达。生产毒素时的主要挑战是获得具有正确二硫键模式的正确折叠的蛋白质，以确保感兴趣毒素的活性。本综述介绍了可用于在细菌、酵母、昆虫细胞或哺乳动物细胞中表达毒素的策略，以及不涉及细胞的合成方法，如无细胞生物合成和肽合成。同时概述了这些不同系统生产毒素的主要优点和缺点，并讨论了在处理具有高生物活性的蛋白质时需要考虑的生物安全问题。
