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.

Keywords: animal toxins; bioinsecticide; heterologous expression; neurotoxin; recombinant protein expression; recombinant toxins; toxin-inspired drug; venom.

FIGURE 1

Comparison of Myotoxin II from Bothrops asper, β-bungarotoxin from Bungarus multicinctus and a phospholipase A₂ (PLA₂) from Naja nigricollis. They are homologous proteins that cluster together due to sequence similarity and share an archetypical PLA₂ fold. Nevertheless, they differ in their toxic activity.

FIGURE 2

Distribution of PTMs among toxins listed in Uniprot-Toxprot, the Animal Toxin Annotation Project. Only 12% of the listed animal toxins do not have any described PTMs (A). From the toxins with PTMs, 85% had disulfide bonds (B). After disulfide bonds, glycosylation, and especially N-glycosylation, is the most common PTM described for toxins (C).

FIGURE 3

Summary of the strategies available for the successful expression of toxins in an E. coli bacterial expression system.

FIGURE 4

Summary of the strategies available and their advantages for the expression of correctly folded toxins in a P. pastoris expression system.

FIGURE 5

Summary of the strategies available for the expression of correctly folded toxins in a baculovirus insect cell expression system.

FIGURE 6

Summary of the strategies available and their main advantages for the expression of functional toxins in a mammalian cell expression system.

FIGURE 7

Representation of how venom gland organoids are derived from snake venom gland cells. The cells isolated from the snake venom glands (A) are cultured as organoids (B) that secrete venom (yellow) containing active toxins (spheres), which can be isolated from the organoids (C).

FIGURE 8

Schematic representation of a cell-free protein synthesis (CFPS) system. Cell culture extract is supplemented with essential reagents for protein synthesis. Upon addition of a nucleic acid template coding for the toxin of interest, the toxin gene is transcribed and translated into a toxin that might need assisted folding.

FIGURE 9

Schematic representation of a solid-phase peptide synthesis (SPPS) system. The solid-phase (resin) is activated for peptide synthesis upon deprotection of the reactive amino group (A). The amino acids are added sequentially on the C-terminal, while remaining protected on the N-terminal. The incoming amino acid forms a peptide bond with the free N-terminal on the resin (B). Deprotection of the amino acid linked to the resin leaves a free amino group ready to react with the next N-protected amino acid (C). The cycle is repeated until all the amino acids are incorporated, upon which the peptide is cleaved from the resin and refolded in vitro (D).

FIGURE 10

Representation of the discovery of broadly-neutralizing antibodies using consensus toxins. Consensus toxins are designed toxins that represent an average sequence of a collection of homologous toxins (A). By using consensus toxins in phage display selection campaigns (B), selected antibodies might be able to neutralize not only the consensus toxin, but also the natural toxins used in the consensus toxin design.