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.

Cloning and soluble expression of mature α-luffin from Luffa cylindrica inE. coli using SUMO fusion protein

“…Escherichia coli has been the workhorse of gene expression for many years, but unfortunately cytoplasmic expression of many recombinant toxins mostly has led to inclusion bodies formation, requiring comprehensive in vitro refolding steps to access their biological activities (Balduino et al, 2011;Lyukmanova et al, 2016;Shimokawa-Falcao et al, 2017). On the other hand, the refolding process is not cost effective in many cases; therefore enhancing protein solubility by other strategies is a better approach to obtain high production levels (Lee et al, 2008).…”

“…Reducing proteolytic degradation of the recombinant protein and simplifying the purification process are other advantages of SUMO fusion technology. Recombinant protein expression in E. coli, using SUMO, has increased the yield of difficult-toexpress proteins (Shimokawa-Falcao et al, 2017).…”

Expression of soluble native protein in Escherichia coli using a cold-shock SUMO tag-fused expression vector

“…Previous study showed that the solubilizing tags (glutathione S -transferase (GST), small ubiquitin-like modifier protein (SUMO), and maltose-binding protein (MBP), etc. ) could efficiently decrease the formation of inclusion bodies within the soluble proteins production [ 12 ], in the majority of the cases, although the tags require immediate removal for further applications. Notably, in genetically engineered pharmaceutical proteins, the final forms of the purified proteins must exert a native protein function [ 10 ].…”

“…The enzymes enterokinase, thrombin, factor Xa, tobacco etch virus (TEV) protease and Ulp1 are commonly applied to remove the fusion tags [ 13 ]. Among these proteases, Ulp1 protease that recognizes only SUMO tertiary structure and leaves the POI with its native N-terminal [ 12 ]. However, although a variety of techniques can be used to produce recombinant proteins, a universal method for the preparation of soluble, homogeneous and native N-terminus target proteins is not available to date.…”

Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein

“…Since the expression of protein in its soluble format is more desirable considering no need for further refolding steps different approaches have been applied to address this problem ( 26 ). Reducing rate of protein synthesis by decreased inducer concentration, induction at lower temperatures ( 27 ), medium optimization ( 28 ), co-expression of chaperons ( 29 ) and foldases ( 30 ) and the use of fusion tags ( 31 ) have been investigated in several studies.…”