Four disulfide-bridged scorpion beta neurotoxin CssII: Heterologous expression and proper folding in vitro

Abstract

The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and an FXa proteolytic cleavage region. This recombinant vector was transfected into E. coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/L of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9,392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9,400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7,538.6 Da as expected. Since native CssII is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na v 1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.

Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

“…4a). Because Css2, Css4, and Cn2 recognize Na v 1.6 channels (6,8), it is highly likely that these toxins interact with a similar region of Na ϩ channels and that Css2, Css4, and Cn2 share a similar region for the binding to Na v 1.6 channels (see supplemental Fig. S5).…”

“…The most noxious and abundant molecule found in the venom of this scorpion is Css2 (LD 50 of 0.7 g/20 g of mice of the strain CD1) (6). In addition, the Mexican scorpion Centruroides noxius Hoffmann produces toxin Cn2, one of the most abundant and noxious peptides against mammals (LD 50 of 0.25 g/20 g of mice of the strain CD1) (7).…”

“…Sequence comparison shows that Cn2 presents a high similarity with other ␤-toxins from C. suffusus suffusus, such as Css2 and Css4 (around 90%, see below). Both toxins, Cn2 and Css2, are specific to Na ϩ channel subtype Na v 1.6 (6,8).…”

In vitro refolding and functional analysis of polyhistidine-tagged Buthus martensii Karsch antitumor-analgesic peptide produced in Escherichia coli

“…Shao et al (2013) found co-expression of Bmk toxin peptides with thioredoxin A generated a less-reducing cytoplasmic environment that promoted disulfide bond formation. Many toxin polypeptides rich in disulfide bridges, such as scorpion toxin CssII, Cn5, snake toxin WTX, and bothropstoxin-1, have been refolded from inclusion bodies by in vitro oxidative renaturation (Altamirano et al 1999;Balduino et al 2011;Estrada et al 2007;Lyukmanova et al 2009). …”

Expressão heteróloga e caracterização de duas toxinas do escorpião Tityus serrulatus

“…A escolha do sistema de expressão em P. pastoris pode garantir o sucesso da produção de toxinas recombinantes, uma vez que esse sistema é capaz de realizar o enovelamento correto do peptídeo, com a formação das pontes dissulfeto, para assegurar que a atividade biológica seja preservada (ESCOUBAS et al, 2003;ESTRADA et al, 2007). No nosso laboratório, diversas toxinas já foram expressas nesse sistema com sucesso, como a serinoprotease da serpente Crotalus durissus collilineatus, a Ts19 Frag-II e a hialuronidase do escorpião T. serrulatus (BOLDRINI-FRANCA et al, 2015).…”