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Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen’s formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.
Paula A. L. Calabria; Lhiri Hanna A. L. Shimokawa-Falcão; Monica Colombini; Ana M. Moura-Da-Silva; Katia C. Barbaro; Eliana L. Faquim-Mauro; Geraldo S. Magalhaes. Design and Production of a Recombinant Hybrid Toxin to Raise Protective Antibodies against Loxosceles Spider Venom. Toxins 2019, 11, 108 .
AMA StylePaula A. L. Calabria, Lhiri Hanna A. L. Shimokawa-Falcão, Monica Colombini, Ana M. Moura-Da-Silva, Katia C. Barbaro, Eliana L. Faquim-Mauro, Geraldo S. Magalhaes. Design and Production of a Recombinant Hybrid Toxin to Raise Protective Antibodies against Loxosceles Spider Venom. Toxins. 2019; 11 (2):108.
Chicago/Turabian StylePaula A. L. Calabria; Lhiri Hanna A. L. Shimokawa-Falcão; Monica Colombini; Ana M. Moura-Da-Silva; Katia C. Barbaro; Eliana L. Faquim-Mauro; Geraldo S. Magalhaes. 2019. "Design and Production of a Recombinant Hybrid Toxin to Raise Protective Antibodies against Loxosceles Spider Venom." Toxins 11, no. 2: 108.
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.
Lhiri H. A. L. Shimokawa-Falcão; Maria C. Caporrino; Katia C. Barbaro; Maisa S. Della-Casa; Geraldo S. Magalhães. Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria. Toxins 2017, 9, 82 .
AMA StyleLhiri H. A. L. Shimokawa-Falcão, Maria C. Caporrino, Katia C. Barbaro, Maisa S. Della-Casa, Geraldo S. Magalhães. Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria. Toxins. 2017; 9 (3):82.
Chicago/Turabian StyleLhiri H. A. L. Shimokawa-Falcão; Maria C. Caporrino; Katia C. Barbaro; Maisa S. Della-Casa; Geraldo S. Magalhães. 2017. "Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria." Toxins 9, no. 3: 82.