Expression and purification of recombinant alpha-toxin AnCra1 from the scorpion Androctonus crassicauda and its functional characterization on mammalian sodium channels

Abstract

Background Alpha-scorpion toxins with long-chain peptide and four disulfide bonds represent diverse pharmacological profiles for various subtypes of voltage-gated sodium channels. Obtaining the natural toxins are difficult and time-consuming process, which represents the major difficulty to interpreting analysis of their structural and functional properties. Methods and ResultsThis study describes the toxin peptide and plasmid construct containing the gene coding for mammalian toxin AnCra1 from the scorpion Androctonus crassicauda venom. We have established genetic construction of fusion protein in pET32a + vector containing thioredoxin (Trx-tag), enterokinase cleavage site and 6xhistidine-tag for efficient expression in Escherichia coli strain RG2 (DE3). The soluble expressed peptide, then purified by Ni-NTA resin affinity chromatography and its purity was confirmed by reverse-phase HPLC and mass spectrometry (7433.54 Da.). The electrophysiological data showed that recombinant AnCra1 selectively inhibits the fast inactivation of hNav1.7 channel (EC 50 = 136.7 ± 6.6 nM). Conclusions Our findings demonstrate that the AnCra1 is structurally and functionally analogous to alpha excitatory toxins; furthermore, expression and purification of bioactive scorpion toxins in bacterial cells can be a practicable and efficient way to obtain a novel source of toxin peptides as tools to study the function and physiological responses of ion channels.

Expression of Recombinant Stonustoxin Alpha Subunit and Preparation of polyclonal antiserum for Stonustoxin Neutralization Studies

“…The lower expression yields in the case of recombinant toxins compared to other recombinant proteins can be attributed to their toxic nature. As an example, Bayatzadeh et al expressed the alpha subunit of the scorpion Androctonus crassicauda venom in E. coli RG2 (DE3), with a nal yield of only 1 mg/L [47].…”

Scorpion Venom as a Source of Antimicrobial Peptides: Overview of Biomolecule Separation, Analysis and Characterization Methods

“…For example, in order to circumvent the low yield of production for the recombinant insect-selective neurotoxin BjαIT from Hottentotta judaicus , a study resorted to applying affinity chromatography using a nitriloacetic (Ni-NTA) agarose column as the purification method, highlighting the high efficiency of this technique for purification of the desired neurotoxin responsible for an insecticidal effect [ 73 ]. The same goes for recombinant α-toxin AnCra1, purified from Androctonus crassicauda , where affinity chromatography was used for optimal purification [ 74 ]. To purify a recombinant GST fusion protein containing a long-chain potassium scorpion toxin from Mesobuthus martensii (BmTXKβ), a GST resin (glutathione-sepharose 4B) was used for affinity chromatography.…”