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Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria.

ABSTRACT: Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.

SUBMITTER: Shimokawa-Falcao LH

PROVIDER: S-EPMC5371837 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria.

Shimokawa-Falcão Lhiri H A L LH Caporrino Maria C MC Barbaro Katia C KC Della-Casa Maisa S MS Magalhães Geraldo S GS Magalhães Geraldo S GS

Toxins 20170227 3

Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into <i>Escherichia c ...[more]

PMID: 28264436

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Project description:BACKGROUND:Heterologous gene expression is well established for various prokaryotic model systems. However, low yield, incorrect folding and instability still impede the production of soluble, bioactive proteins. To improve protein production with the Gram-positive host Bacillus subtilis, a secretory expression system was designed that enhances translocation, folding and stability of heterologous proteins, and simplifies purification. Based on the theta-replication plasmid pHT01, a B. subtilis secretory expression vector was constructed that encodes a fusion protein consisting of a signal peptide and a StrepII-tag linked to a SUMO-tag serving as a folding catalyst. The gene of a protein of interest can be translationally fused to the SUMO cassette and an additional 6xHis-tag encoding region. In order to maximize secretory expression of the construct by fitting the signal peptide to the StrepII-SUMO part of the fusion protein, a B. subtilis signal-peptide library was screened with the Escherichia coli alkaline phosphatase PhoA as a reporter. RESULTS:The YoaW signal peptide-encoding region (SPyoaW) was identified with highest secretory expression capacity in context with the StrepII-SUMO-tag fusion in a B. subtilis eightfold extracellular protease deletion strain. PhoA activity and fusion protein production was elevated by a factor of approximately five when compared to an ?-amylase (AmyQ) signal peptide construct. Replacement of PhoA with a single-chain variable fragment antibody specific for GFP or the B. amyloliquefaciens RNase barnase, respectively, resulted in a similar enhancement of secretory expression, demonstrating universality of the YoaW signal peptide-StrepII-SUMO encoding cassette for secretory expression in B. subtilis. Optimisation of codon usage and culture conditions further increased GFP-specific scFv fusion-protein production, and a simple affinity purification strategy from culture supernatant with removal of the StrepII-SUMO-tag by SenP-processing yielded 4 mg of pure, soluble and active GFP-specific scFv from 1 l of culture under standard laboratory conditions. CONCLUSIONS:The new expression system employing a YoaW signal peptide-StrepII-SUMO fusion will simplify secretory protein production and purification with B. subtilis. It can obviate the need for time consuming individual signal-peptide fitting to maximize yield for many different heterologous proteins of interest.

Project description:Snakebite is classified as a priority Neglected Tropical Disease by the World Health Organization. Understanding the pathology of individual snake venom toxins is of great importance when developing more effective snakebite therapies. Snake venoms may induce a range of pathologies, including haemolytic activity. Although snake venom-induced erythrocyte lysis is not the primary cause of mortality, haemolytic activity can greatly debilitate victims and contributes to systemic haemotoxicity. Current assays designed for studying haemolytic activity are not suitable for rapid screening of large numbers of toxic compounds. Consequently, in this study, a high-throughput haemolytic assay was developed that allows profiling of erythrocyte lysis, and was validated using venom from a number of medically important snake species (Calloselasma rhodostoma, Daboia russelii, Naja mossambica, Naja nigricollis and Naja pallida). The assay was developed in a format enabling direct integration into nanofractionation analytics, which involves liquid chromatographic separation of venom followed by high-resolution fractionation and subsequent bioassaying (and optional proteomics analysis), and parallel mass spectrometric detection. Analysis of the five snake venoms via this nanofractionation approach involving haemolytic assaying provided venom-cytotoxicity profiles and enabled identification of the toxins responsible for haemolytic activity. Our results show that the elapid snake venoms (Naja spp.) contained both direct and indirect lytic toxins, while the viperid venoms (C. rhodostoma and D. russelii) only showed indirect lytic activities, which required the addition of phospholipids to exert cytotoxicity on erythrocytes. The haemolytic venom toxins identified were mainly phospholipase A2s and cytotoxic three finger toxins. Finally, the applicability of this new analytical method was demonstrated using a conventional snakebite antivenom treatment and a small-molecule drug candidate to assess neutralisation of venom cytotoxins.

Project description:Despite the unparalleled diversity of venomous snakes in Australia, research has concentrated on a handful of medically significant species and even of these very few toxins have been fully sequenced. In this study, venom gland transcriptomes were sequenced from eleven species of small Australian elapid snakes, from eleven genera, spanning a broad phylogenetic range. The particularly large number of sequences obtained for three-finger toxin (3FTx) peptides allowed for robust reconstructions of their dynamic molecular evolutionary histories. We demonstrated that each species preferentially favoured different types of ?-neurotoxic 3FTx, probably as a result of differing feeding ecologies. The three forms of ?-neurotoxin [Type I (also known as (aka): short-chain), Type II (aka: long-chain) and Type III] not only adopted differential rates of evolution, but have also conserved a diversity of residues, presumably to potentiate prey-specific toxicity. Despite these differences, the different ?-neurotoxin types were shown to accumulate mutations in similar regions of the protein, largely in the loops and structurally unimportant regions, highlighting the significant role of focal mutagenesis. We theorize that this phenomenon not only affects toxin potency or specificity, but also generates necessary variation for preventing/delaying prey animals from acquiring venom-resistance. This study also recovered the first full-length sequences for multimeric phospholipase A2 (PLA2) 'taipoxin/paradoxin' subunits from non-Oxyuranus species, confirming the early recruitment of this extremely potent neurotoxin complex to the venom arsenal of Australian elapid snakes. We also recovered the first natriuretic peptides from an elapid that lack the derived C-terminal tail and resemble the plesiotypic form (ancestral character state) found in viper venoms. This provides supporting evidence for a single early recruitment of natriuretic peptides into snake venoms. Novel forms of kunitz and waprin peptides were recovered, including dual domain kunitz-kunitz precursors and the first kunitz-waprin hybrid precursors from elapid snakes. The novel sequences recovered in this study reveal that the huge diversity of unstudied venomous Australian snakes are of considerable interest not only for the investigation of venom and whole organism evolution but also represent an untapped bioresource in the search for novel compounds for use in drug design and development.

Dataset Information

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria.

Publications

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria.

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