Unveiling Tst3, a Multi-Target Gating Modifier Scorpion α Toxin from Tityus stigmurus Venom of Northeast Brazil: Evaluation and Comparison with Well-Studied Ts3 Toxin of Tityus serrulatus.

Studies on the interaction sites of peptide toxins and ion channels typically involve site-directed mutations in toxins. However, natural mutant toxins exist among them, offering insights into how the evolutionary process has conserved crucial sequences for activities and molecular target selection. In this study, we present a comparative investigation using electrophysiological approaches and computational analysis between two alpha toxins from evolutionarily close scorpion species of the genus Tityus, namely, Tst3 and Ts3 from T. stigmurus and T. serrulatus, respectively. These toxins exhibit three natural substitutions near the C-terminal region, which is directly involved in the interaction between alpha toxins and Nav channels. Additionally, we characterized the activity of the Tst3 toxin on Nav1.1-Nav1.7 channels. The three natural changes between the toxins did not alter sensitivity to Nav1.4, maintaining similar intensities regarding their ability to alter opening probabilities, delay fast inactivation, and induce persistent currents. Computational analysis demonstrated a preference for the down conformation of VSD4 and a shift in the conformational equilibrium towards this state. This illustrates that the sequence of these toxins retained the necessary information, even with alterations in the interaction site region. Through electrophysiological and computational analyses, screening of the Tst3 toxin on sodium isoform revealed its classification as a classic α-NaTx with a broad spectrum of activity. It effectively delays fast inactivation across all tested isoforms. Structural analysis of molecular energetics at the interface of the VSD4-Tst3 complex further confirmed this effect.

A robust genome assembly with transcriptomic data from the striped bark scorpion, Centruroides vittatus.

Scorpions, a seemingly primitive, stinging arthropod taxa, are known to exhibit marked diversity in their venom components. These venoms are known for their human pathology, but they are also important as models for therapeutic and drug development applications. In this study, we report a high-quality genome assembly and annotation of the striped bark scorpion, Centruroides vittatus, created with several shotgun libraries. The final assembly is 760 Mb in size, with a BUSCO score of 97.8%, a 30.85% GC, and an N50 of 2.35 Mb. We estimated 36,189 proteins with 37.32% assigned to Gene Ontology (GO) terms in our GO annotation analysis. We mapped venom toxin genes to 18 contigs and 2 scaffolds. We were also able to identify expression differences between venom gland (telson) and body tissue (carapace) with 19 sodium toxin and 14 potassium toxin genes to 18 contigs and 2 scaffolds. This assembly, along with our transcriptomic data, provides further data to investigate scorpion venom genomics.

Expression and characterization of scFv-6009FV in Pichia pastoris with improved ability to neutralize the neurotoxin Cn2 from Centruroides noxius.

Small single-chain variable fragments (scFv) are promising biomolecules to inhibit and neutralize toxins and to act as antivenoms. In this work, we aimed to produce a functional scFv-6009FV in the yeast Pichia pastoris, which inhibits the pure Cn2 neurotoxin and the whole venom of Centruroides noxius. We were able to achieve yields of up to 31.6 ± 2 mg/L in flasks. Furthermore, the protein showed a structure of 6.1 % α-helix, 49.1 % ß-sheet, and 44.8 % of random coil by CD. Mass spectrometry confirmed the amino acid sequence and showed no glycosylation profile for this molecule. Purified scFv-6009FV allowed us to develop anti-scFvs in rabbits, which were then used in affinity columns to purify other scFvs. Determination of its half-maximal inhibitory concentration value (IC50) was 40 % better than the scFvs produced by E. coli as a control. Finally, we found that scFv-6009FV was able to inhibit ex vivo the pure Cn2 toxin and the whole venom from C. noxius in murine rescue experiments. These results demonstrated that under the conditions assayed here, P. pastoris is suited to produce scFv-6009FV that, compared to scFvs produced by E. coli, maintains the characteristics of an antibody and neutralizes the Cn2 toxin more effectively.

The scorpion toxin BeKm-1 blocks hERG cardiac potassium channels using an indispensable arginine residue.

BeKm-1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (Kv11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm-1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double-mutant cycle analysis data, to obtain an in silico model of the hERG-BeKm-1 complex. Adding to the previous mutagenesis study of BeKm-1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm-1R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts.

Divergence in toxin antigenicity and venom enzymes in Tityus melici, a medically important scorpion, despite transcriptomic and phylogenetic affinities with problematic Brazilian species.

The Brazilian scorpion Tityus melici, native to Minas Gerais and Bahia, is morphologically related to Tityus serrulatus, the most medically significant species in Brazil. Despite inhabiting scorpion-envenomation endemic regions, T. melici venom remains unexplored. This work evaluates T. melici venom composition and function using transcriptomics, enzymatic activities, and in vivo and in vitro immunological analyses. Next-Generation Sequencing unveiled 86 components putatively involved in venom toxicity: 39 toxins, 28 metalloproteases, seven disulfide isomerases, six hyaluronidases, three phospholipases and three amidating enzymes. T. serrulatus showed the highest number of toxin matches with 80-100 % sequence similarity. T. melici is of medical importance as it has a venom LD50 of 0.85 mg/kg in mice. We demonstrated venom phospholipase A2 activity, and elevated hyaluronidase and metalloprotease activities compared to T. serrulatus, paralleling our transcriptomic findings. Comparison of transcriptional levels for T. serrulatus and T. melici venom metalloenzymes suggests species-specific expression patterns in Tityus. Despite close phylogenetic association with T. serrulatus inferred from COI sequences and toxin similarities, partial neutralization of T. melici venom toxicity was achieved when using the anti-T. serrulatus antivenom, implying antigenic divergence among their toxins. We suggest that the Brazilian therapeutic scorpion antivenom could be improved to effectively neutralize T. melici venom.

Genome-wide identification, structural homology analysis, and evolutionary diversification of the phospholipase D gene family in the venom gland of three scorpion species.

BACKGROUND: Venom phospholipase D (PLDs), dermonecrotic toxins like, are the major molecules in the crude venom of scorpions, which are mainly responsible for lethality and dermonecrotic lesions during scorpion envenoming. The purpose of this study was fivefold: First, to identify transcripts coding for venom PLDs by transcriptomic analysis of the venom glands from Androctonus crassicauda, Hottentotta saulcyi, and Hemiscorpius lepturus; second, to classify them by sequence similarity to known PLDs and motif extraction method; third, to characterize scorpion PLDs; fourth to structural homology analysis with known dermonecrotic toxins; and fifth to investigate phylogenetic relationships of the PLD proteins. RESULTS: We found that the venom gland of scorpions encodes two PLD isoforms: PLD1 ScoTox-beta and PLD2 ScoTox-alpha I. Two highly conserved regions shared by all PLD1s beta are GAN and HPCDC (HX2PCDC), and the most important conserved regions shared by all PLD2s alpha are two copies of the HKDG (HxKx4Dx6G) motif. We found that PLD1 beta is a 31-43 kDa acidic protein containing signal sequences, and PLD2 alpha is a 128 kDa basic protein without known signal sequences. The gene structures of PLD1 beta and PLD2 alpha contain 6 and 21 exons, respectively. Significant structural homology and similarities were found between the modeled PLD1 ScoTox-beta and the crystal structure of dermonecrotic toxins from Loxosceles intermedia. CONCLUSIONS: This is the first report on identifying PLDs from A. crassicauda and H. saulcyi venom glands. Our work provides valuable insights into the diversity of scorpion PLD genes and could be helpful in future studies on recombinant antivenoms production.

Identification, Characterization, and Modeling of a Bioinsecticide Protein Isolated from Scorpion Venom gland: A Three-Finger Protein.

Background: The majority of insecticides target sodium channels. The increasing emergence of resistance to the current insecticides has persuaded researchers to search for alternative compounds. Scorpion venom gland as a reservoir of peptides or proteins, which selectively target insect sodium channels. These proteins would be an appropriate source for finding new suitable anti-insect components. Methods: Transcriptome of venom gland of scorpion Mesobuthus eupeus was obtained by RNA extraction and complementary DNA library synthesis. The obtained transcriptome was blasted against protein databases to find insect toxins against sodium channel based on the statistically significant similarity in sequence. Physicochemical properties of the identified protein were calculated using bioinformatics software. The three-dimensional structure of this protein was determined using homology modeling, and the final structure was assessed by molecular dynamics simulation. Results: The sodium channel blocker found in the transcriptome of M. eupeus venom gland was submitted to the GenBank under the name of meuNa10, a stable hydrophilic protein consisting of 69 amino acids, with the molecular weight of 7721.77 g/mol and pI of 8.7. The tertiary structure of meuNa10 revealed a conserved LCN-type cysteine-stabilized alpha/beta domain stabilized by eight cysteine residues. The meuNa10 is a member of the 3FP superfamily consisting of three finger-like beta strands. Conclusion: This study identified meuNa10 as a small insect sodium channel-interacting protein with some physicochemical properties, including stability and water-solubility, which make it a good candidate for further in vivo and in vitro experiments in order to develop a new bioinsecticide.

Production of recombinant scorpion antivenoms in E. coli: current state and perspectives.

Scorpion envenomation is a serious health problem in tropical and subtropical zones. The access to scorpion antivenom is sometimes limited in availability and specificity. The classical production process is cumbersome, from the hyper-immunization of the horses to the IgG digestion and purification of the F(ab)'2 antibody fragments. The production of recombinant antibody fragments in Escherichia coli is a popular trend due to the ability of this microbial host to produce correctly folded proteins. Small recombinant antibody fragments, such as single-chain variable fragments (scFv) and nanobodies (VHH), have been constructed to recognize and neutralize the neurotoxins responsible for the envenomation symptoms in humans. They are the focus of interest of the most recent studies and are proposed as potentially new generation of pharmaceuticals for their use in immunotherapy against scorpion stings of the Buthidae family. This literature review comprises the current status on the scorpion antivenom market and the analyses of cross-reactivity of commercial scorpion anti-serum against non-specific scorpion venoms. Recent studies on the production of new recombinant scFv and nanobodies will be presented, with a focus on the Androctonus and Centruroides scorpion species. Protein engineering-based technology could be the key to obtaining the next generation of therapeutics capable of neutralizing and cross-reacting against several types of scorpion venoms. KEY POINTS: • Commercial antivenoms consist of predominantly purified equine F(ab)'2fragments. • Nanobody-based antivenom can neutralize Androctonus venoms and have a low immunogenicity. • Affinity maturation and directed evolution are used to obtain potent scFv families against Centruroides scorpions.

A first molecular characterization of the scorpion telson microbiota of Hadrurus arizonensis and Smeringurus mesaensis.

Scorpions represent an ancient lineage of arachnids that have radiated across the globe and are incredibly resilient-since some thrive in harsh environments and can exist on minimal and intermittent feedings. Given the emerging importance of microbiomes to an organism's health, it is intriguing to suggest that the long-term success of the scorpion bauplan may be linked to the microbiome. Little is known about scorpion microbiomes, and what is known, concentrates on the gut. The microbiome is not limited to the gut, rather it can be found within tissues, fluids and on external surfaces. We tested whether the scorpion telson, the venom-producing organ, of two species, Smeringurus mesaensis and Hadrurus arizonensis, contain bacteria. We isolated telson DNA from each species, amplified bacterial 16S rRNA genes, and identified the collection of bacteria present within each scorpion species. Our results show for the first time that telsons of non-buthid scorpion species do indeed contain bacteria. Interestingly, each scorpion species has a phylogenetically unique telson microbiome including Mollicutes symbionts. This study may change how we view scorpion biology and their venoms.

An overview of Tityus cisandinus scorpion venom: Transcriptome and mass fingerprinting reveal conserved toxin homologs across the Amazon region and novel lipolytic components.

Tityus cisandinus, a neglected medically important scorpion in Ecuadorian and Peruvian Amazonia, belongs to a complex of species related to the eastern Amazon endemic Tityus obscurus, spanning a distribution of ca. 4000 km. Despite high morbidity and mortality rates, no effective scorpion antivenom is currently available in the Amazon region. Knowledge of the structural/functional relationships between T. cisandinus venom components and those from related Amazonian species is crucial for designing region-specific therapeutic antivenoms. In this work, we carried out the first venom gland transcriptomic study of an Amazonian scorpion outside Brazil, T. cisandinus. We also fingerprinted its total venom through MALDI-TOF MS, which supported our transcriptomic findings. We identified and calculated the expression level of 94 components: 60 toxins, 25 metalloproteases, five disulfide isomerases, three amidating enzymes, one hyaluronidase, and also uncovered transcripts encoding novel lipolytic beta subunits produced by New World buthid scorpions. This study demonstrates the high similarity between T. cisandinus and T. obscurus venoms, reinforcing the existence of a neglected complex of genetically and toxinologically related Amazonian scorpions of medical importance. Finally, we demonstrated the low recognition of currently available therapeutic sera against T. cisandinus and T. obscurus venoms, and concluded that these should be improved to protect against envenomation by Amazonian Tityus spp.

Differential venom gland gene expression analysis of juvenile and adult scorpions Androctonus crassicauda.

BACKGROUND: The Androctonus crassicauda, belonging to the genus Androctonus of the family Buthidae, is the most venomous scorpion in Middle East countries. However, the venom gland transcriptome profile of A. crassicauda scorpion has not yet been studied. In this study, we elucidated and compared the venom gland gene expression profiles of adult and juvenile male scorpion A. crassicauda using high-throughput transcriptome sequencing. This is the first report of transcriptional analysis of the venom glands of scorpions in different growth stages, with insights into the identification of the key genes during venom gland development. RESULTS: A total of 209,951 mRNA transcripts were identified from total RNA-seq data, of which 963 transcripts were differentially expressed (DE) in adult and juvenile scorpions (p < 0.01). Overall, we identified 558 up-regulated and 405 down-regulated transcripts in the adult compared to the juvenile scorpions, of which 397 and 269 unique unigenes were annotated, respectively. GO and KEGG enrichment analyses indicated that the metabolic, thermogenesis, cytoskeleton, estrogen signaling, GnRH signaling, growth hormone signaling, and melanogenesis pathways were affected by two different growth conditions and the results suggested that the DE genes related to those pathways are important genes associated with scorpion venom gland development, in which they may be important in future studies, including Chs, Elovl, MYH, RDX, ACTN, VCL, PIP5K, PP1C, FGFR, GNAS, EGFR, CREB, CoA, PLCB, CALM, CACNA, PKA and CAMK genes. CONCLUSIONS: These findings broadened our knowledge of the differences between adult and juvenile scorpion venom and opened new perspectives on the application of comparative transcriptome analysis to identify the special key genes.

Recombinantly expressed MeICT, a new toxin from Mesobuthus eupeus scorpion, inhibits glioma cell proliferation and downregulates Annexin A2 and FOXM1 genes.

Gliomas are highly invasive and lethal malignancy that do not respond to current therapeutic approaches. Novel therapeutic agents are required to target molecular mechanisms involved in glioma progression. MeICT is a new short-chain toxin isolated from Mesobuthus eupeus scorpion venom. This toxin contained 34 amino acid residues and belongs to chloride channels toxins. In this study, the coding sequence of MeICT was cloned into the pET32Rh vector and a high yield of soluble recombinant MeICT was expressed and purified. Recombinant MeICT-His significantly inhibited the proliferation and migration of glioma cells at low concentration. In vivo studies showed that MeICT was not toxic when administrated to mice at high doses. We also determined the effect of MeICT on the mRNA expression of MMP-2, Annexin A2 and FOXM-2 that are key molecules in the progression and invasion of glioma. Expression of Annexin A2 and FOXM1 mRNA was significantly down-regulated following treatment with MeICT. However, no significant decrease in the expression of MMP-2 gene was identified. In this study a short toxin with four disulfide bonds was successfully produced and its anti-cancer effects was detected. Our findings suggest that recombinant MeICT can be considered as a new potent agent for glioma targeting.

Phylogenomics of Scorpions Reveal Contemporaneous Diversification of Scorpion Mammalian Predators and Mammal-Active Sodium Channel Toxins.

Scorpions constitute a charismatic lineage of arthropods and comprise more than 2500 described species. Found throughout various tropical and temperate habitats, these predatory arachnids have a long evolutionary history, with a fossil record that began in the Silurian. While all scorpions are venomous, the asymmetrically diverse family Buthidae harbors nearly half the diversity of extant scorpions, and all but one of the 58 species that are medically significant to humans. However, the lack of a densely sampled scorpion phylogeny has hindered broader inferences of the diversification dynamics of scorpion toxins. To redress this gap, we assembled a phylogenomic data set of 100 scorpion venom gland transcriptomes and genomes, emphasizing the sampling of highly toxic buthid genera. To infer divergence times of venom gene families, we applied a phylogenomic node dating approach for the species tree in tandem with phylostratigraphic bracketing to estimate the minimum ages of mammal-specific toxins. Our analyses establish a robustly supported phylogeny of scorpions, particularly with regard to relationships between medically significant taxa. Analysis of venom gene families shows that mammal-active sodium channel toxins (NaTx) have independently evolved in five lineages within Buthidae. Temporal windows of mammal-targeting toxin origins are correlated with the basal diversification of major scorpion mammal predators such as shrews, bats, and rodents. These results suggest an evolutionary model of relatively recent diversification of buthid NaTx homologs in response to the diversification of scorpion predators. [Adaptation; arachnids; phylogenomic dating; phylostratigraphy; venom.].

Genomic Structure of Two Kv1.3 Channel Blockers from Scorpion Mesobuthus eupeus and Sea Anemone Stichodactyla haddoni and Construction of their Chimeric Peptide as a Novel Blocker.

Different toxins acting on Kv1.3 channel have been isolated from animal venom. MeuKTX toxin from Mesobuthus eupeus phillipsi scorpion and shtx-k toxin from Stichodactyla haddoni sea anemone have been identified as two effective Kv1.3 channel blockers. In this work, we characterized the genomic organization of both toxins. MeuKTX gene contains one intron and two exons, similar to the most scorpion toxins. There are a few reports of genomic structure of sea anemone toxins acting on Kv channels. The sequence encoding mature peptide of shtx-k was located in an exon separated by an intron from the coding exon of the propeptide and signal region. In order to make a peptide with more affinity for Kv1.3 channel and greater stability, the shtx-k/ MeuKTX chimeric peptide was designed and constructed using splicing by overlap extension-PCR (SOE-PCR) method. MeuKTX, shtx-k, and shtx-k/MeuKTX were cloned and the expression of the soluble proteins in E. coli was determined. Molecular docking studies indicated more inhibitory effect of shtx-k/MeuKTX on Kv1.3 channel compared to shtx-k and MeuKTX toxins. Key amino acids binding channel from both toxins, also involved in interaction of chimeric peptide with channel. Our results showed that the fusion peptide, shtx-k/MeuKTX could be an effective agent to target Kv1.3 channel.

Reduced Toxicity of Centruroides vittatus (Say, 1821) May Result from Lowered Sodium ß Toxin Gene Expression and Toxin Protein Production.

Body tissue and venom glands from an eastern population of the scorpion Centruroides vittatus (Say, 1821) were homogenized and molecular constituents removed to characterize putative sodium ß toxin gene diversity, RT-qPCR, transcriptomic, and proteomic variation. We cloned sodium ß toxins from genomic DNA, conducted RT-qPCR experiments with seven sodium ß toxin variants, performed venom gland tissue RNA-seq, and isolated venom proteins for mass spectrophotometry. We identified >70 putative novel sodium ß toxin genes, 111 toxin gene transcripts, 24 different toxin proteins, and quantified sodium ß toxin gene expression variation among individuals and between sexes. Our analyses contribute to the growing evidence that venom toxicity among scorpion taxa and their populations may be associated with toxin gene diversity, specific toxin transcripts variation, and subsequent protein production. Here, slight transcript variation among toxin gene variants may contribute to the major toxin protein variation in individual scorpion venom composition.

A non-lethal method for studying scorpion venom gland transcriptomes, with a review of potentially suitable taxa to which it can be applied.

Scorpion venoms are mixtures of proteins, peptides and small molecular compounds with high specificity for ion channels and are therefore considered to be promising candidates in the venoms-to-drugs pipeline. Transcriptomes are important tools for studying the composition and expression of scorpion venom. Unfortunately, studying the venom gland transcriptome traditionally requires sacrificing the animal and therefore is always a single snapshot in time. This paper describes a new way of generating a scorpion venom gland transcriptome without sacrificing the animal, thereby allowing the study of the transcriptome at various time points within a single individual. By comparing these venom-derived transcriptomes to the traditional whole-telson transcriptomes we show that the relative expression levels of the major toxin classes are similar. We further performed a multi-day extraction using our proposed method to show the possibility of doing a multiple time point transcriptome analysis. This allows for the study of patterns of toxin gene activation over time a single individual, and allows assessment of the effects of diet, season and other factors that are known or likely to influence intraindividual venom composition. We discuss the gland characteristics that may allow this method to be successful in scorpions and provide a review of other venomous taxa to which this method may potentially be successfully applied.

Expression and purification of recombinant alpha-toxin AnCra1 from the scorpion Androctonus crassicauda and its functional characterization on mammalian sodium channels.

BACKGROUND: Alpha-scorpion toxins with long-chain peptide and four disulfide bonds represent diverse pharmacological profiles for various subtypes of voltage-gated sodium channels. Obtaining the natural toxins are difficult and time-consuming process, which represents the major difficulty to interpreting analysis of their structural and functional properties. METHODS AND RESULTS: This study describes the toxin peptide and plasmid construct containing the gene coding for mammalian toxin AnCra1 from the scorpion Androctonus crassicauda venom. We have established genetic construction of fusion protein in pET32a + vector containing thioredoxin (Trx-tag), enterokinase cleavage site and 6xhistidine-tag for efficient expression in Escherichia coli strain RG2 (DE3). The soluble expressed peptide, then purified by Ni-NTA resin affinity chromatography and its purity was confirmed by reverse-phase HPLC and mass spectrometry (7433.54 Da.). The electrophysiological data showed that recombinant AnCra1 selectively inhibits the fast inactivation of hNav1.7 channel (EC50 = 136.7 ± 6.6 nM). CONCLUSIONS: Our findings demonstrate that the AnCra1 is structurally and functionally analogous to alpha excitatory toxins; furthermore, expression and purification of bioactive scorpion toxins in bacterial cells can be a practicable and efficient way to obtain a novel source of toxin peptides as tools to study the function and physiological responses of ion channels.

Mapping the interaction surface of scorpion ß-toxins with an insect sodium channel.

The interaction of insect-selective scorpion depressant ß-toxins (LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus) with the Blattella germanica sodium channel, BgNav1-1a, was investigated using site-directed mutagenesis, electrophysiological analyses, and structural modeling. Focusing on the pharmacologically defined binding site-4 of scorpion ß-toxins at the voltage-sensing domain II (VSD-II), we found that charge neutralization of D802 in VSD-II greatly enhanced the channel sensitivity to Lqh-dprIT3. This was consistent with the high sensitivity of the splice variant BgNav2-1, bearing G802, to Lqh-dprIT3, and low sensitivity of BgNav2-1 mutant, G802D, to the toxin. Further mutational and electrophysiological analyses revealed that the sensitivity of the WT = D802E < D802G < D802A < D802K channel mutants to Lqh-dprIT3 correlated with the depolarizing shifts of activation in toxin-free channels. However, the sensitivity of single mutants involving IIS4 basic residues (K4E = WT << R1E < R2E < R3E) or double mutants (D802K = K4E/D802K = R3E/D802K > R2E/D802K > R1E/D802K > WT) did not correlate with the activation shifts. Using the cryo-EM structure of the Periplaneta americana channel, NavPaS, as a template and the crystal structure of LqhIT2, we constructed structural models of LqhIT2 and Lqh-dprIT3-c in complex with BgNav1-1a. These models along with the mutational analysis suggest that depressant toxins approach the salt-bridge between R1 and D802 at VSD-II to form contacts with linkers IIS1-S2, IIS3-S4, IIIP5-P1 and IIIP2-S6. Elimination of this salt-bridge enables deeper penetration of the toxin into a VSD-II gorge to form new contacts with the channel, leading to increased channel sensitivity to Lqh-dprIT3.

Overexpression and purification of a toxic peptide LaIT2 from Japanese scorpion, Liocheles australasiae.

In Japan, there are two species of scorpions, Madara scorpion (Isometrus maculatus) and Yaeyama scorpion (Liocheles australasiae), and both of them are living in Yaeyama island. It has been shown that Liocheles australasiae has venom including ß-toxin acting on K+-channels (ß-KTx) (Juichi et al., 2018) [1]. Interestingly, LaIT2, one of the toxins found in the venom of Liocheles australasiae, displays the virulence for insects but almost not for mammals. Until now, molecular mechanism of the functional specificity of LaIT2 is unknown. To clear this issue, we tried to establish the overexpression system of LaIT2 in Rosetta-gami B (DE3) pLysS, which have trxB/gor mutations to induce the disulfide bond formation. In this study, we have succeeded to overexpress the recombinant LaIT2 (rLaIT2) as a thioredoxin (Trx)-tagged protein, and established the purification protocol with Ni2+-NTA column chromatography, enterokinase digestion, and HPLC. We succeeded to obtain approximately 0.5 mg of rLaIT2 from the E. coli cells cultured in 1 L of M9 culture medium. Intramolecular disulfide bonding pattern of rLaIT2 was identified by endopeptidase fragmentation and mass spectrometry. rLaIT2 showed insecticidal activity and antimicrobial activity, and these are almost identical to those of natural LaIT2. 1H-15N HSQC spectrum of 15N-labeled rLaIT2 indicated that the rLaIT2 has a stable conformation.