Strategies for Heterologous Expression, Synthesis, and Purification of Animal Venom Toxins

Abstract

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.

Identification of a sensory neuron Cav2.3 inhibitor within a new superfamily of macro-conotoxins

“…At a general level, this study illustrates that a combination of data mining and recombinant expression in E. coli can pave the way for a detailed analysis of structural and functional features of newly identified, macro-conotoxins. We propose that with the advent of E. coli expression systems such as csCyDisCo and DisCoTune (25,28,56), as well as others (57, 58), the time is ripe to begin the systematic exploration of a new realm of macro-conotoxins. These efforts will help provide a better understanding of the biological correlates of having large venom components, with the overarching aim of connecting the biochemical and molecular characteristics of venom components with the biology and behavior of cone snails.…”

De novo designed proteins neutralize lethal snake venom toxins

“…First, de novo protein design does not rely on animal immunization, and yields proteins that can be manufactured using recombinant DNA technology, thereby creating a source for continuous production of product with limited batch-to-batch variation. Second, computational design enables the creation of binding proteins with high affinity and specificity without needing extensive experimental screening programs that often rely on pure toxins, which can be challenging to isolate from whole venoms or generate via recombinant expression 32,33 . Third, the small size of designed proteins could offer enhanced tissue penetration 34 compared to large antibodies, enabling rapid toxin neutralization and thereby potentially be particularly effective in neutralizing non-systemic toxicities, such as those leading to local tissue damage.…”

CysPresso: a classification model utilizing deep learning protein representations to predict recombinant expression of cysteine-dense peptides

“…One of the challenges of using CDPs for therapeutic purposes is that they are difficult to synthesize and express. While the disulfide bonds are important for the stability of the molecule, incorrect linkage can lead to misfolded non-native isomers [ 14 – 16 ]. Recent work using high-throughput expression systems to express CDPs in mammalian cells has allowed the screening of hundreds of CDPs for expression [ 17 , 18 ].…”