TY - JOUR

T1 - Strategies for Heterologous Expression, Synthesis, and Purification of Animal Venom Toxins

AU - Rivera-de-Torre, Esperanza

AU - Rimbault, Charlotte

AU - Jenkins, Timothy P.

AU - Sørensen, Christoffer V.

AU - Damsbo, Anna

AU - Saez, Natalie J.

AU - Duhoo, Yoan

AU - Hackney, Celeste Menuet

AU - Ellgaard, Lars

AU - Laustsen, Andreas H.

PY - 2022

Y1 - 2022

N2 - 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.

AB - 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.

KW - animal toxins

KW - venom

KW - neurotoxin

KW - heterologous expression

KW - recombinant toxins

KW - recombinant protein expression

KW - bioinsecticide

KW - toxin-inspired drug

KW - FREE PROTEIN-SYNTHESIS

KW - RECOMBINANT FUSION PROTEIN

KW - LARGE-SCALE PRODUCTION

KW - UNNATURAL AMINO-ACIDS

KW - HIGH-LEVEL EXPRESSION

KW - THROMBIN-LIKE ENZYME

KW - ESCHERICHIA-COLI

KW - PICHIA-PASTORIS

KW - ION-CHANNEL

KW - FUNCTIONAL EXPRESSION

U2 - 10.3389/fbioe.2021.811905

DO - 10.3389/fbioe.2021.811905

M3 - Review

C2 - 35127675

VL - 9

JO - Frontiers in Bioengineering and Biotechnology

JF - Frontiers in Bioengineering and Biotechnology

SN - 2296-4185

M1 - 811905

ER -