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Toxins 2017, 9(3), 82; doi:10.3390/toxins9030082

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

Laboratory of Immunopathology, Butantan Institute, Av. Vital Brazil 1500, 05503-900 São Paulo, SP, Brazil
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Academic Editor: Michel Dugon
Received: 11 January 2017 / Revised: 16 February 2017 / Accepted: 22 February 2017 / Published: 27 February 2017
(This article belongs to the Section Animal Venoms)
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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. View Full-Text
Keywords: construction vector; venom toxin; phospholipase D; Loxosceles gaucho; SUMO tag; disintegrins Ulp1 protease; snake toxin; spider toxin; protein expression construction vector; venom toxin; phospholipase D; Loxosceles gaucho; SUMO tag; disintegrins Ulp1 protease; snake toxin; spider toxin; protein expression
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Shimokawa-Falcão, L.H.A.L.; Caporrino, M.C.; Barbaro, K.C.; Della-Casa, M.S.; Magalhães, G.S. 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.

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