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The three-fingered toxin family and more precisely short-chain α-neurotoxins (also known as Type I α-neurotoxins) are crucial in defining the elapid envenomation process, but paradoxically, they are barely neutralized by current elapid snake antivenoms. This work has been focused on the primary structural identity among Type I neurotoxins in order to create a consensus short-chain α-neurotoxin with conserved characteristics. A multiple sequence alignment considering the twelve most toxic short-chain α-neurotoxins reported from the venoms of the elapid genera Acanthophis, Oxyuranus, Walterinnesia, Naja, Dendroaspis and Micrurus led us to propose a short-chain consensus α-neurotoxin, here named ScNtx. The synthetic ScNtx gene was de novo constructed and cloned into the expression vector pQE30 containing a 6His-Tag and an FXa proteolytic cleavage region. Escherichia coli Origami cells transfected with the pQE30/ScNtx vector expressed the recombinant consensus neurotoxin in a soluble form with a yield of 1.5 mg/L of culture medium. The 60-amino acid residue ScNtx contains canonical structural motifs similar to α-neurotoxins from African elapids and its LD 50 of 3.8 µg/mice is similar to the most toxic short-chain α-neurotoxins reported from elapid venoms. Furthermore, ScNtx was also able to antagonize muscular, but not neuronal, nicotinic acetylcholine receptors (nAChR). Rabbits immunized with ScNtx were able to immune-recognize short-chain α-neurotoxins within whole elapid venoms. Type I neurotoxins are difficult to isolate and purify from natural sources; therefore, the heterologous expression of molecules such ScNtx, bearing crucial motifs and key amino acids, is a step forward to create common immunogens for developing cost-effective antivenoms with a wider spectrum of efficacy, quality and strong therapeutic value.

Crotoxin complex CA/CB and crotamine are the main toxins associated with Crotalus envenomation besides the enzymatic activities of phospholipases (PLA2) and proteases. The neutralization at least of the crotoxin complex by neutralizing the subunit B could be a key in the production process of antivenoms against crotalids. Therefore, in this work, a Crotoxin B was recombinantly expressed to evaluate its capacity as an immunogen and its ability to produce neutralizing antibodies against crotalid venoms. A Crotoxin B transcript from Crotalus tzabcan was cloned into a pCR®2.1-TOPO vector (Invitrogen, Waltham, MA, USA) and subsequently expressed heterologously in bacteria. HisrCrotoxin B was extracted from inclusion bodies and refolded in vitro. The secondary structure of HisrCrotoxin B was comparable to the secondary structure of the native Crotoxin B, and it has PLA2 activity as the native Crotoxin B. HisrCrotoxin B was used to immunize rabbits, and the obtained antibodies partially inhibited the activity of PLA2 from C. tzabcan. The anti-HisrCrotoxin B antibodies neutralized the native Crotoxin B and the whole venoms from C. tzabcan, C. s. salvini, and C. mictlantecuhtli. Additionally, anti-HisrCrotoxin B antibodies recognized native Crotoxin B from different Crotalus species, and they could discriminate venom in species with high or low levels of or absence of Crotoxin B.

Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, which acting by themselves, or in synergism, are the cause of the envenomation symptoms and death. Here, two mRNA transcripts, one that codes for a metalloprotease and another for a serine-protease, were isolated from a Bothrops ammodytoides venom gland. The metalloprotease and serine-protease transcripts were cloned on a pCR®2.1-TOPO vector and consequently expressed in a recombinant way in E. coli (strains Origami and M15, respectively), using pQE30 vectors. The recombinant proteins were named rBamSP_1 and rBamMP_1, and they were formed by an N-terminal fusion protein of 16 amino acid residues, followed by the sequence of the mature proteins. After bacterial expression, each recombinant enzyme was recovered from inclusion bodies and treated with chaotropic agents. The experimental molecular masses for rBamSP_1 and rBamMP_1 agreed with their expected theoretical ones, and their secondary structure spectra obtained by circular dichroism were comparable to that of similar proteins. Additionally, equivalent mixtures of rBamSP_1, rBamMP_1 together with a previous reported recombinant phospholipase, rBamPLA2_1, were used to immunize rabbits to produce serum antibodies, which in turn recognized serine-proteases, metalloproteases and PLA2s from B. ammodytoides and other regional viper venoms. Finally, rabbit antibodies neutralized the 3LD50 of B. ammodytoides venom.

The transcriptome of the venom glands of the Phoneutria depilata spider was analyzed using RNA-seq with an Illumina protocol, which yielded 86,424 assembled transcripts. A total of 682 transcripts were identified as potentially coding for venom components. Most of the transcripts found were neurotoxins (156) that commonly act on sodium and calcium channels. Nevertheless, transcripts coding for some enzymes (239), growth factors (48), clotting factors (6), and a diuretic hormone (1) were found, which have not been described in this spider genus. Furthermore, an enzymatic characterization of the venom of P. depilata was performed, and the proteomic analysis showed a correlation between active protein bands and protein sequences found in the transcriptome. The transcriptomic analysis of P. depilata venom glands show a deeper description of its protein components, allowing the identification of novel molecules that could lead to the treatment of human diseases, or could be models for developing bioinsecticides.

The antimicrobial and immunomodulatory capacities of the peptide Css54 and the chemokine MCP-1 were tested. The first, a peptide isolated from the venom of the scorpion Centruroides suffusus suffusus was synthesized chemically. In contrast, the second is a monocyte chemoattractant expressed as a recombinant protein in our lab. It was observed in vitro that Css54 inhibited the growth of Salmonella enterica serovar Typhimurium (6.2 µg/mL). At high concentrations, it was toxic to macrophages (25 µg/mL), activated macrophage phagocytosis (1.5 µg/mL), and bound Salmonella LPS (3 µg/mL). On the other hand, the recombinant MCP-1 neither inhibited the growth of Salmonella Typhimurium nor was it toxic to macrophages (up to 25 µg/mL), nor activated macrophage phagocytosis or bound Salmonella LPS (up to 3 µg/mL). Although it was observed in vivo in mice Balb/C that both Css54 and MCP-1 did not resolve the intraperitoneal infection by S. Typhimurium, Css54 decreased the expression of IL-6 and increased IL-10, IL-12p70, and TNF-α levels; meanwhile, MCP-1 decreased the expression of IFN-γ and increased IL-12p70 and TNF-α. It was also observed that the combination of both molecules Css54 and MCP-1 increased the expression of IL-10 and TNF-α.

An mRNA transcript that codes for a Disintegrin and a Metalloprotease from a venom gland of the viper Bothrops ammodytoides was isolated. Vector pCR®2.1-TOPO was used to clone the Disintegrin transcript and then subcloned in the pET-28a vector to express the protein in the E. coli strain BL21. The recombinant disintegrin, HisrDisintegrin, comprises 38 residues at the N-terminal and 75 residues of the Disintegrin, including 14 cysteines or 7 disulfide bonds. HisrDisintegrin was obtained soluble in the intracellular fraction and separated by affinity chromatography. The experimental molecular mass of HisrDisintegrin, 11,750.8 Da, agreed with its theoretical value, including the cleavage of a Met residue caused by a bacterial post-translational modification. HisrDisintegrin was folded in in vitro conditions and then purified, finding a main fraction with a comparable molecular mass. The HisrDisintegrin contains an alpha/beta structure, as observed by circular dichroism. Its biological activity demonstrated the growth reduction of the human endothelial (HMEC-1) cells and the human mammary gland adenocarcinoma (MDA-MB-231). Furthermore, HisrDisintegrin inhibits the adhesion of HMEC-1 and MDA-MB-231 cells to the proteins of the extracellular matrix (laminin (LN), fibronectin (FN), and vitronectin (VN)).

Sonicated protein isolates were recovered from Chenopodium quinua, Phaseoulus vulgaris and Lens culinaris to develop a functional matrix by assessing the physicochemical and functional properties. The plant protein isolates were prepared from powdered materials followed by sonication in alkaline medium using a Box-Behnken design. pH (6–10), a buffer-to-material ratio (5:1 to 15:1) and sonication time (0–20 min) were taken as independent variables, whereas protein yield was taken as the dependent variable. A pH of 9, 20 min treatment, and a buffer-to-material ratio of 5:1 were the optimal extraction conditions for quinoa and black beans, whereas a 1:10 ratio was suitable for lentils. Sonication in alkaline medium caused partial protein unfolding and these isolates; in turn, the molecular weight affected the emulsifying activity and stability. Moreover, sonication had a strong effect on the gelation temperature, emulsifying activity, the water, and oil sorption. Sonication improved protein yield and exposed amino acids such as glutamic acid, aspartic acid, leucine and glycine. In turn, thiol groups were responsible for the increased in gelation temperature. The better gelling property coupled with high emulsifying property of these proteins show potential application as protein emulsifiers in the production of gels, sausages, and pet foods.

δ-Atracotoxins, also known as δ-hexatoxins, are spider neurotoxic peptides, lethal to both vertebrates and insects. Their mechanism of action involves the binding to of the S3/S4 loop of the domain IV of the voltage-gated sodium channels (Nav). Because of the chemical difficulties of synthesizing folded synthetic δ-atracotoxins correctly, here we explore an expression system that is designed to produce biologically active recombinant δ-atracotoxins, and a number of variants, in order to establish certain amino acids implicated in the pharmacophore of this lethal neurotoxin. In order to elucidate and verify which amino acid residues play a key role that is toxic to vertebrates and insects, amino acid substitutes were produced by aligning the primary structures of several lethal δ-atracotoxins with those of δ-atracotoxins-Hv1b; a member of the δ-atracotoxin family that has low impact on vertebrates and is not toxic to insects. Our findings corroborate that the substitutions of the amino acid residue Y22 from δ-atracotoxin-Mg1a (Magi4) to K22 in δ-atracotoxin-Hv1b reduces its mammalian activity. Moreover, the substitutions of the amino acid residues Y22 and N26 from δ-atracotoxin-Mg1a (Magi4) to K22 and N26 in δ-atracotoxin-Hv1b reduces its insecticidal activity. Also, the basic residues K4 and R5 are important for keeping such insecticidal activity. Structural models suggest that such residues are clustered onto two bioactive surfaces, which share similar areas, previously reported as bioactive surfaces for scorpion α-toxins. Furthermore, these bioactive surfaces were also found to be similar to those found in related spider and anemone toxins, which affect the same Nav receptor, indicating that these motifs are important not only for scorpion but may be also for animal toxins that affect the S3/S4 loop of the domain IV of the Nav.

Spider venoms include various peptide toxins that modify the ion currents, mainly of excitable insect cells. Consequently, scientific research on spider venoms has revealed a broad range of peptide toxins with different pharmacological properties, even for mammal species. In this work, thirty animal venoms were screened against hK v 1.5, a potential target for atrial fibrillation therapy. The whole venom of the spider Oculicosa supermirabilis , which is also insecticidal to house crickets, caused voltage-gated potassium ion channel modulation in hK v 1.5. Therefore, a peptide from the spider O. supermirabilis venom, named Osu1, was identified through HPLC reverse-phase fractionation. Osu1 displayed similar biological properties as the whole venom; so, the primary sequence of Osu1 was elucidated by both of N-terminal degradation and endoproteolytic cleavage. Based on its primary structure, a gene that codifies for Osu1 was constructed de novo from protein to DNA by reverse translation. A recombinant Osu1 was expressed using a pQE30 vector inside the E. coli SHuffle expression system. recombinant Osu1 had voltage-gated potassium ion channel modulation of human hK v 1.5, and it was also as insecticidal as the native toxin. Due to its novel primary structure, and hypothesized disulfide pairing motif, Osu1 may represent a new family of spider toxins.

This study aimed to assess the thermal stability of the bioactive compounds from annatto seed extract, encapsulated by ionic gelation using quinoa proteins, lentil proteins, soy proteins, and sodium caseinate as carrying materials. The 10.0% aqueous dispersions of the different proteins (carriers) were prepared and mixed with the annatto seed extract. The dispersions were then extruded into a calcium chloride solution to induce the extract encapsulation. The capsules were characterized by encapsulation efficiency, particle size, infrared transmission spectroscopy, confocal microscopy, and scanning electron microscopy (SEM). The antioxidant and antimicrobial activities, the polyphenol compounds, and bixin content from the free and encapsulated extract were assessed once stored for 12 d at different temperatures (4 °C, 25 °C, and 65 °C). The results demonstrated the ability of the proteins to encapsulate the annatto extract with encapsulation efficiencies ranging from 58% to 80%, where the protein structure and amino acid content were the relevant factors to obtain high encapsulation efficiencies. The free extracts stored at 65 °C for 12 d experienced a degradation of bixin and polyphenol compounds, respectively. Conversely, the encapsulated extract had degradations from ~34.00% to ~4.05% for polyphenol compounds and ~20.0% for bixin, respectively. These proteins have a potential encapsulation capacity of annatto extract by ionic gelation.