Mohammad Ali Bayatzadeh1, Abbas Zare Mirakabadi2, Nahid Babaei1, Abdolhassan Doulah3, Abbas Doosti4. 1. Department of Molecular Cell Biology and Genetics, Bushehr Branch, Islamic Azad University, Bushehr, Iran. 2. Venomous Animals and Antivenom Production Department, Razi Vaccine and Serum Research Institute, Agricultural Research- Education and Extension Organization, Hesarak, Karaj, Alborz, Iran. a.zare@rvsri.ac.ir. 3. Department of Nursing, Ahvaz Branch, Islamic Azad University, Ahvaz, Iran. 4. Biotechnology Research Center, Islamic Azad University, Shahrekord, Iran.
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 RESULTS: This 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 (EC50 = 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.
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 RESULTS: This 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 (EC50 = 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.
Authors: V Quintero-Hernández; E Ortiz; M Rendón-Anaya; E F Schwartz; B Becerril; G Corzo; L D Possani Journal: Toxicon Date: 2011-09-28 Impact factor: 3.033