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

Lhiri Shimokawa-Falcão,Maria Caporrino,Maisa Della-Casa,Katia Barbaro,Geraldo Magalhães

doi:10.3390/toxins9030082

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.

Highlights

Venoms are essentially used for defense and/or prey immobilization, they represent a natural reservoir of several biologically active molecules that might become a valuable tool for the development of new biotechnological and pharmaceutical products [1,2,3,4]
Using a specially designed expression vector, we demonstrate below the successful expression of both a novel spider phospholipase D (PLD) and a snake disintegrin into E. coli
In order to remove the small ubiquitin-related modifier (SUMO) from the protein of interest (POI), the sequence of Ulp1 protease under control of lacUV5 promoter was inserted into the cassette

Summary

Introduction

Venoms are essentially used for defense and/or prey immobilization, they represent a natural reservoir of several biologically active molecules that might become a valuable tool for the development of new biotechnological and pharmaceutical products [1,2,3,4]. Understanding how toxins work will assist in the development of better treatment for human envenomation [5]. Obtaining enough toxins from the original source is one of the major challenges in this field. To overcome this problem, many toxins genes have been expressed into heterologous organisms to obtain sufficient material to perform functional, structural, and biochemical characterization. As spiders produce a limited amount of venom, this system has been of extreme value to study interesting toxins with antiarrhythmic [8], antimicrobial [9], analgesic [10], insecticidal [11]

Methods

Results

Conclusion