Hi1a Improves Sensorimotor Deficit following Endothelin-1-Induced Stroke in Rats but Does Not Improve Functional Outcomes following Filament-Induced Stroke in Mice.

Activation of acid-sensing ion channel 1a (ASIC1a) plays a major role in mediating acidosis-induced neuronal injury following a stroke. Therefore, the inhibition of ASIC1a is a potential therapeutic avenue for the treatment of stroke. Venom-peptide Hi1a, a selective and highly potent ASIC1a inhibitor, reduces the infarct size and functional deficits when injected into the brain after stroke in rodents. However, its efficacy when administered using a clinically relevant route of administration remains to be established. Therefore, the current investigation aims to examine the efficacy of systemically administered Hi1a, using two different models of stroke in different species. Mice were subjected to the filament model of middle cerebral artery occlusion (MCAO) and treated with Hi1a systemically using either a single- or multiple-dosing regimen. 24 h poststroke, mice underwent functional testing, and the brain infarct size was assessed. Rats were subjected to endothelin-1 (ET-1)-induced MCAO and treated with Hi1a intravenously 2 h poststroke. Rats underwent functional tests prior to and for 3 days poststroke, when infarct volume was assessed. Mice receiving Hi1a did not show any improvements in functional outcomes, despite a trend toward reduced infarct size. This trend for reduced infarct size in mice was consistent regardless of the dosing regimen. There was also a trend toward lower infarct size in rats treated with Hi1a. More specifically, Hi1a reduced the amount of damage occurring within the somatosensory cortex, which was associated with an improved sensorimotor function in Hi1a-treated rats. Thus, this study suggests that Hi1a or more brain-permeable ASIC1a inhibitors are a potential stroke treatment.

Evaluation of Peptide Ligation Strategies for the Synthesis of the Bivalent Acid-Sensing Ion Channel Inhibitor Hi1a.

Hi1a is a naturally occurring bivalent spider-venom peptide that is being investigated as a promising molecule for limiting ischemic damage in strokes, myocardial infarction, and organ transplantation. However, the challenges associated with the synthesis and production of the peptide in large quantities have slowed the progress in this area; hence, access to synthetic Hi1a is an essential milestone for the development of Hi1a as a pharmacological tool and potential therapeutic.

Genetic or Pharmacological Ablation of Acid-Sensing Ion Channel 1a (ASIC1a) Is Not Neuroprotective in a Mouse Model of Spinal Cord Injury.

Acid-sensing ion channel 1a (ASIC1a) is a proton-activated channel that is expressed ubiquitously throughout the central nervous system and in various types of immune cells. Its role in spinal cord injury (SCI) is controversial; inhibition of ASIC1a has been reported to improve SCI pathology in vivo, but conversely, gene ablation increased kainite-mediated excitotoxic cell death in vitro. Here, we re-examined the role of ASIC1a in a mouse model of SCI. First, we observed functional outcomes up to 42 days post-operation (DPO) in SCI mice with a selective genetic ablation of ASIC1a. Mice lacking ASIC1a had significantly worsened locomotor ability and increased lesion size compared with mice possessing the ASIC1a gene. Next, we explored pharmacological antagonism of this ion channel by administering the potent ASIC1a inhibitor, Hi1a. Consistent with a role for ASIC1a to attenuate excitotoxicity, accelerated neuronal cell loss was found at the lesion site in SCI mice treated with Hi1a, but there were no differences in locomotor recovery. Moreover, ASIC1a inhibition did not cause significant alterations to neutrophil migration, microglial density, or blood-spinal cord barrier integrity.

A peptide toxin in ant venom mimics vertebrate EGF-like hormones to cause long-lasting hypersensitivity in mammals.

Venoms are excellent model systems for studying evolutionary processes associated with predator-prey interactions. Here, we present the discovery of a peptide toxin, MIITX2-Mg1a, which is a major component of the venom of the Australian giant red bull ant Myrmecia gulosa and has evolved to mimic, both structurally and functionally, vertebrate epidermal growth factor (EGF) peptide hormones. We show that Mg1a is a potent agonist of the mammalian EGF receptor ErbB1, and that intraplantar injection in mice causes long-lasting hypersensitivity of the injected paw. These data reveal a previously undescribed venom mode of action, highlight a role for ErbB receptors in mammalian pain signaling, and provide an example of molecular mimicry driven by defensive selection pressure.

Total Synthesis of the Spider-Venom Peptide Hi1a.

Hi1a is a venom peptide from the Australian funnel-web spider Hadronyche infensa with a complex tertiary structure. Hi1a has neuroprotective and cardioprotective properties due to its potent inhibition of acid-sensing ion channel 1a (ASIC1a) and is currently being pursued as a novel therapy for acute ischemic events. Herein, we describe the total synthesis of Hi1a using native chemical ligation. The synthetic peptide was successfully folded and exhibited similar inhibitory activity on ASIC1a to recombinant Hi1a.

Therapeutic Inhibition of Acid-Sensing Ion Channel 1a Recovers Heart Function After Ischemia-Reperfusion Injury.

BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.

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

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.

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists.

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαß structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.

High-Throughput Production of Oxidized Animal Toxins in Escherichia coli.

High-throughput production (HTP) of synthetic genes is becoming an important tool to explore the biological function of the extensive genomic and meta-genomic information currently available from various sources. One such source is animal venom, which contains thousands of novel bioactive peptides with potential uses as novel therapeutics to treat a plethora of diseases as well as in environmentally benign bioinsecticide formulations. Here, we describe a HTP platform for recombinant bacterial production of oxidized disulfide-rich proteins and peptides from animal venoms. High-throughput, host-optimized, gene synthesis and subcloning, combined with robust HTP expression and purification protocols, generate a semiautomated pipeline for the accelerated production of proteins and peptides identified from genomic or transcriptomic libraries. The platform has been applied to the production of thousands of animal venom peptide toxins for the purposes of drug discovery, but has the power to be universally applicable for high-level production of various and diverse target proteins in soluble form. This chapter details the HTP protocol for gene synthesis and production, which supported high levels of peptide expression in the E. coli periplasm using a cleavable DsbC fusion. Finally, target proteins and peptides are purified using automated HTP methods, before undergoing quality control and screening.

The antitrypanosomal diarylamidines, diminazene and pentamidine, show anthelmintic activity against Haemonchus contortus in vitro.

Parasitic nematodes pose a major threat to livestock production worldwide. The blood-feeding parasite Haemonchus contortus is a key small-ruminant pathogen that causes anaemia, and thereby seriously impacts animal health and production. Control of this parasite relies largely upon broad-spectrum anthelmintics, but new drugs are urgently needed to combat the threat of widespread multidrug resistance. Repurposing drugs can accelerate the development pipeline by reducing costs and risks, and can be an effective way of quickly bringing new antiparasitic drugs to market. Diarylamidine compounds such as pentamidine and diminazene have been employed in the treatment of trypanosomiasis and leishmaniasis in both human and veterinary settings, but their activity against parasitic worms has not yet been reported. We screened a small panel of diarylamidine compounds against H. contortus to assess their potential to be repurposed as anthelmintic drugs. Pentamidine and diminazene inhibited H. contortus larval development at low micromolar concentrations (IC50 4.9 µM and 16.1 µM, respectively, in a drug-susceptible isolate) with no existing cross-resistance in two multidrug resistant isolates and a monepantel-resistant isolate. Combinations of pentamidine with commercial anthelmintics showed additive activity, with no significant synergism detected. Pentamidine and diminazene showed different life-stage patterns of activity; both were active against early stage larvae in development assays, but only diminazene was active against the infective L3 stage in migration assays. This suggests some differences in uptake of the two drugs across the nematode cuticle, or differences in the nature and expression patterns of their molecular targets. As pentamidine and diminazene have been reported to be potent inhibitors of mammalian acid-sensing ion channels (ASIC), we tested the activity of known ASIC inhibitors against H. contortus to probe whether these channels may represent potential anthelmintic targets in nematodes. Remarkably, the spider-venom peptide Hi1a, a potent inhibitor of ASIC1a, inhibited H. contortus larval development with an IC50 of 22.9 ± 1.9 µM. This study highlights the potential use of diarylamidines as anthelmintics, although their activity needs to be confirmed in vivo. In addition, our demonstration that ASIC inhibitors have anthelmintic activity raises the possibility that this family of ion channels may represent a novel anthelmintic target.

The modulation of acid-sensing ion channel 1 by PcTx1 is pH-, subtype- and species-dependent: Importance of interactions at the channel subunit interface and potential for engineering selective analogues.

Acid-sensing ion channels (ASICs) are primary acid sensors in the mammalian nervous system that are activated by protons under conditions of local acidosis. They have been implicated in a range of pathologies including ischemic stroke (ASIC1a subtype) and peripheral pain (ASIC1b and ASIC3). Although the spider venom peptide PcTx1 is the best-studied ASIC modulator and is neuroprotective in rodent models of ischemic stroke, little experimental work has been done to examine its molecular interaction with human ASIC1a or the off-target ASIC1b. The complementary face of the acidic pocket binding site of PcTx1 is where these channels differ in sequence. We show here that although PcTx1 is 10-fold less potent at human ASIC1a than the rat channel, the apparent affinity for the two channels is comparable. We examined the pharmacophore of PcTx1 for human ASIC1a and rat ASIC1b, and show that inhibitory and stimulatory effects at each ASIC1 variant is driven mostly by a shared set of core peptide pharmacophore residues that bind to the thumb domain, while peptide residues that interact with the complementary face of the biding site underlie species and subtype-dependent differences in activity that may allow manipulation of ASIC1 variant selectivity. Finally, the stimulatory effect of PcTx1 on rat ASIC1a when applied under mildly alkaline pH correlates with low receptor occupancy. These new insights into the interactions between PcTx1 with ASIC1 subtypes demonstrates the complexity of its mechanism of action, and highlights important implications to consider when using PcTx1 as a pharmacological tool to study ASIC function.

Versatile spider venom peptides and their medical and agricultural applications.

Spiders have been evolving complex and diverse repertoires of peptides in their venoms with vast pharmacological activities for more than 300 million years. Spiders use their venoms for prey capture and defense, hence they contain peptides that target both prey (mainly arthropods) and predators (other arthropods or vertebrates). This includes peptides that potently and selectively modulate a range of targets such as ion channels, receptors and signaling pathways involved in physiological processes. The contribution of these targets in particular disease pathophysiologies makes spider venoms a valuable source of peptides with potential therapeutic use. In addition, peptides with insecticidal activities, used for prey capture, can be exploited for the development of novel bioinsecticides for agricultural use. Although we have already reviewed potential applications of spider venom peptides as therapeutics (in 2010) and as bioinsecticides (in 2012), a considerable number of research articles on both topics have been published since, warranting an updated review. Here we explore the most recent research on the use of spider venom peptides for both medical and agricultural applications.

ArachnoServer 3.0: an online resource for automated discovery, analysis and annotation of spider toxins.

Summary: ArachnoServer is a manually curated database that consolidates information on the sequence, structure, function and pharmacology of spider-venom toxins. Although spider venoms are complex chemical arsenals, the primary constituents are small disulfide-bridged peptides that target neuronal ion channels and receptors. Due to their high potency and selectivity, these peptides have been developed as pharmacological tools, bioinsecticides and drug leads. A new version of ArachnoServer (v3.0) has been developed that includes a bioinformatics pipeline for automated detection and analysis of peptide toxin transcripts in assembled venom-gland transcriptomes. ArachnoServer v3.0 was updated with the latest sequence, structure and functional data, the search-by-mass feature has been enhanced, and toxin cards provide additional information about each mature toxin. Availability and implementation: http://arachnoserver.org. Contact: support@arachnoserver.org. Supplementary information: Supplementary data are available at Bioinformatics online.

Inhibition of acid-sensing ion channels by diminazene and APETx2 evoke partial and highly variable antihyperalgesia in a rat model of inflammatory pain.

BACKGROUND AND PURPOSE: Acid-sensing ion channels (ASICs) are primary acid sensors in mammals, with the ASIC1b and ASIC3 subtypes being involved in peripheral nociception. The antiprotozoal drug diminazene is a moderately potent ASIC inhibitor, but its analgesic activity has not been assessed. EXPERIMENTAL APPROACH: We determined the ASIC subtype selectivity of diminazene and the mechanism by which it inhibits ASICs using voltage-clamp electrophysiology of Xenopus oocytes expressing ASICs 1-3. Its peripheral analgesic activity was then assessed relative to APETx2, an ASIC3 inhibitor, and morphine, in a Freund's complete adjuvant (FCA)-induced rat model of inflammatory pain. KEY RESULTS: Diminazene inhibited homomeric rat ASICs with IC50 values of ~200-800 nM, via an open channel and subtype-dependent mechanism. In rats with FCA-induced inflammatory pain in one hindpaw, diminazene and APETx2 evoked more potent peripheral antihyperalgesia than morphine, but the effect was partial for APETx2. APETx2 potentiated rat ASIC1b at concentrations 30-fold to 100-fold higher than the concentration inhibiting ASIC3, which may have implications for its use in in vivo experiments. CONCLUSIONS AND IMPLICATIONS: Diminazene and APETx2 are moderately potent ASIC inhibitors, both inducing peripheral antihyperalgesia in a rat model of chronic inflammatory pain. APETx2 has a more complex ASIC pharmacology, which must be considered when it is used as a supposedly selective ASIC3 inhibitor in vivo. Our use of outbred rats revealed responders and non-responders when ASIC inhibition was used to alleviate inflammatory pain, which is aligned with the concept of number-needed-to-treat in human clinical studies. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

A Strategy for Production of Correctly Folded Disulfide-Rich Peptides in the Periplasm of E. coli.

Recombinant expression of disulfide-reticulated peptides and proteins is often challenging. We describe a method that exploits the periplasmic disulfide-bond forming machinery of Escherichia coli and combines this with a cleavable, solubility-enhancing fusion tag to obtain higher yields of correctly folded target protein than is achievable via cytoplasmic expression. The protocols provided herein cover all aspects of this approach, from vector construction and transformation to purification of the cleaved target protein and subsequent quality control.

High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery.

BACKGROUND: Animal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics. RESULTS: In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in Escherichia coli is described. Here we report the development of a high-throughput automated platform, that could be adapted to the production of other families, to generate the largest ever library of recombinant venom peptides. The peptides were produced in the periplasm of E. coli using redox-active DsbC as a fusion tag, thus allowing the efficient formation of correctly folded disulphide bridges. TEV protease was used to remove fusion tags and recover the animal venom peptides in the native state. Globally, within nine months, out of a total of 4992 synthetic genes encoding a representative diversity of venom peptides, a library containing 2736 recombinant disulphide-reticulated peptides was generated. The data revealed that the animal venom peptides produced in the bacterial host were natively folded and, thus, are putatively biologically active. CONCLUSIONS: Overall this study reveals that high-throughput expression of animal venom peptides in E. coli can generate large libraries of recombinant disulphide-reticulated peptides of remarkable interest for drug discovery programs.

Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli.

BACKGROUND: Animal venoms are large, complex libraries of bioactive, disulphide-rich peptides. These peptides, and their novel biological activities, are of increasing pharmacological and therapeutic importance. However, recombinant expression of venom peptides in Escherichia coli remains difficult due to the significant number of cysteine residues requiring effective post-translational processing. There is also an urgent need to develop high-throughput recombinant protocols applicable to the production of reticulated peptides to enable efficient screening of their drug potential. Here, a comprehensive study was developed to investigate how synthetic gene design, choice of fusion tag, compartment of expression, tag removal conditions and protease recognition site affect levels of solubility of oxidized venom peptides produced in E. coli. RESULTS: The data revealed that expression of venom peptides imposes significant pressure on cysteine codon selection. DsbC was the best fusion tag for venom peptide expression, in particular when the fusion was directed to the bacterial periplasm. While the redox activity of DsbC was not essential to maximize expression of recombinant fusion proteins, redox activity did lead to higher levels of correctly folded target peptides. With the exception of proline, the canonical TEV protease recognition site tolerated all other residues at its C-terminus, confirming that no non-native residues, which might affect activity, need to be incorporated at the N-terminus of recombinant peptides for tag removal. CONCLUSIONS: This study reveals that E. coli is a convenient heterologous host for the expression of soluble and functional venom peptides. Using the optimal construct design, a large and diverse range of animal venom peptides were produced in the µM scale. These results open up new possibilities for the high-throughput production of recombinant disulphide-rich peptides in E. coli.

Molecular dynamics and functional studies define a hot spot of crystal contacts essential for PcTx1 inhibition of acid-sensing ion channel 1a.

BACKGROUND AND PURPOSE: The spider-venom peptide PcTx1 is the most potent and selective inhibitor of acid-sensing ion channel (ASIC) 1a. It has centrally acting analgesic activity and is neuroprotective in rodent models of ischaemic stroke. Understanding the molecular details of the PcTx1 : ASIC1a interaction should facilitate development of therapeutically useful ASIC1a modulators. Previously, we showed that several key pharmacophore residues of PcTx1 reside in a dynamic ß-hairpin loop; conclusions confirmed by recent crystal structures of the complex formed between PcTx1 and chicken ASIC1 (cASIC1). Numerous peptide : channel contacts were observed in these crystal structures, but it remains unclear which of these are functionally important. EXPERIMENTAL APPROACH: We combined molecular dynamics (MD) simulations of the PcTx1 : cASIC1 complex with mutagenesis of PcTx1 and rat ASIC1a. KEY RESULTS: Crystal structures of the PcTx1 : cASIC1 complex indicated that 15 PcTx1 residues form a total of 57 pairwise intermolecular contacts (<5 Å) with 32 channel residues. MD simulations, however, suggested that about half of these interactions do not persist in solution. Mutation to alanine of only eight of 15 PcTx1 contact residues substantially altered ASIC1a inhibition by PcTx1. Our data reveal that many of the peptide-channel interactions observed in the PcTx1 : cASIC1 crystal structures are not important for PcTx1 inhibition of rat ASIC1a. CONCLUSIONS AND IMPLICATIONS: We identified the atomic interactions that are critical for PcTx1 inhibition of ASIC1a. Our data highlight the value of combining structural information, MD and functional experiments to obtain detailed insight into the molecular basis of protein : protein interactions.

High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli.

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.