title: 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 intoEscherichia 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 spiderLoxosceles gauchoand a disintegrin from theBothrops insularissnake. 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.
authors.affiliation: Laboratory of Immunopathology, Butantan Institute, Av. Vital Brazil 1500, 05503-900 São Paulo, SP, Brazil. lhiri.hanna@gmail.com.
authors.forename: Lhiri H A L
authors.initials: LH
authors.lastname: Shimokawa-Falcão
authors.affiliation: Laboratory of Immunopathology, Butantan Institute, Av. Vital Brazil 1500, 05503-900 São Paulo, SP, Brazil. maria.caporrino@butantan.gov.br.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Caporrino
authors.affiliation: Laboratory of Immunopathology, Butantan Institute, Av. Vital Brazil 1500, 05503-900 São Paulo, SP, Brazil. katia.barbaro@butantan.gov.br.
authors.forename: Katia C
authors.initials: KC
authors.lastname: Barbaro
authors.affiliation: Laboratory of Immunopathology, Butantan Institute, Av. Vital Brazil 1500, 05503-900 São Paulo, SP, Brazil. maisa.casa@butantan.gov.br.
authors.forename: Maisa S
authors.initials: MS
authors.lastname: Della-Casa
authors.affiliation: Laboratory of Immunopathology, Butantan Institute, Av. Vital Brazil 1500, 05503-900 São Paulo, SP, Brazil. geraldo.magalhaes@butantan.gov.br.
authors.forename: Geraldo S
authors.initials: GS
authors.lastname: Magalhães
mesh_terms: D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics / Q000633:toxicity; D001792:Blood Platelets / Q000187:drug effects; D017837:Bothrops; D003435:Crotalid Venoms / Q000235:genetics / Q000633:toxicity; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D019483:Disintegrins / Q000235:genetics / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D010739:Phospholipase D / Q000235:genetics / Q000633:toxicity; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000633:toxicity; D011993:Recombinant Fusion Proteins / Q000633:toxicity; D025842:SUMO-1 Protein / Q000235:genetics* / Q000378:metabolism; D013111:Spider Venoms; D013112:Spiders
chemical_list: D060829:Arthropod Proteins; D003435:Crotalid Venoms; D019483:Disintegrins; D010975:Platelet Aggregation Inhibitors; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D013111:Spider Venoms; C557217:insularin, Bothrops insularis; D010739:Phospholipase D; D003546:Cysteine Endopeptidases; C118083:Ulp1 protease
keywords: Loxosceles gaucho; SUMO tag; construction vector; disintegrins Ulp1 protease; phospholipase D; protein expression; snake toxin; spider toxin; venom toxin