Characterization of recombinant alpha‑toxin AnCra1 from Iranian scorpion Androctonus crassicauda venom and its effects on mammalian sodium channels function
Characterization of recombinant alpha‑toxin AnCra1 from Iranian scorpion Androctonus crassicauda venom and its effects on mammalian sodium channels function
Mohammad Ali Bayatzadeh,1,*Abbas Zare Mirakabadi,2
1. Venomous Animals and Antivenom Production Department, Razi Vaccine and Serum Research Institute, Agricultural Research- Education and Extension Organization, Hesarak, Karaj, Alborz, Iran 2. Venomous Animals and Antivenom Production Department, Razi Vaccine and Serum Research Institute, Agricultural Research- Education and Extension Organization, Hesarak, Karaj, Alborz, Iran
Introduction: 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. This study describes the toxin peptide and plasmid construct containing the gene coding for mammalian toxin AnCra1 from the Iranian scorpion Androctonus crassicauda venom.
Methods: 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. Toxicity of recombinant AnCra1 in comparison with native toxin was assayed in NIH mice. The electrophysiological assays of rAnCra1 was examined on hNav1.5 and hNav1.7 channels expressed on HEK293 cell line.
Results: The expression of the TrxA 6 × His -AnCra1 fusion protein was successfully done in RG2 (DE3) and the molecular weight of purified rAnCra1, was determined 7433.54 Da. Recombinant AnCra1 exhibits approximately high toxicity, as reflected by their lethal doses that it was certainly 3.34 ± 0.04 μg per mice. The electrophysiological data showed that rAnCra1 selectively inhibits the fast inactivation of hNav1.7 channel (EC50 = 136.7 ± 6.6 nM)
Conclusion: Our findings demonstrate that the rAnCra1 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.