Expression of a recombinant Phoneutria toxin active in calcium channels

• Published in: Toxicon (Oxford) 2012-10, Vol.60 (5), p.907-918
• Main Authors: Souza, I.A., Cino, E.A., Choy, W.Y., Cordeiro, M.N., Richardson, M., Chavez-Olortegui, C., Gomez, M.V., Prado, M.A.M., Prado, V.F.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analysis of Variance; Animal poisons toxicology. Antivenoms; Animals; Bacterial expression; bioactive properties; Biological and medical sciences; Calcium channel blocker; calcium channels; Calcium Channels - metabolism; Circular Dichroism; Knottin scaffold; Medical sciences; Molecular Sequence Data; Neuropeptides - genetics; Neuropeptides - isolation & purification; Neuropeptides - metabolism; Oligonucleotides - genetics; Phoneutria nigriventer; Plasmids - genetics; Protein Folding; Recombinant Proteins - metabolism; Sequence Analysis, DNA; Spider toxin; Spider Venoms - metabolism; Synaptosomes - metabolism; Toxicology; venoms
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2012.05.026

PnTx3-4 is a toxin isolated from the venom of the spider Phoneutria nigriventer that blocks N-, P/Q-, and R-type voltage-gated calcium channels and has great potential for clinical applications. In this report we used the SUMO system to express large amounts of recombinant PnTx3-4 peptide, which was found in both soluble and insoluble fractions of bacterial extracts. We purified the recombinant toxin from both fractions and showed that the recombinant peptide showed biological activity similar to the native PnTx3-4. In silico analysis of the primary sequence of PnTx3-4 indicated that the peptide conforms to all the criteria of a knottin scaffold. Additionally, circular dichroism spectrum analysis of the recombinant PnTx3-4 predicted that the toxin structure is composed of approximately 53% turns/unordered, 31% α-helix and 16% β-strand, which is consistent with predicted model of the PnTx3-4 knottin scaffold available at the knottin database (http://knottin.cbs.cnrs.fr). These studies provide the basis for future large scale production and structure-function investigation of PnTx3-4. ► Heterologous expression and purification of PnTx3-4, a toxin that blocks N-, P/Q-, and R-type voltage-gated calcium channels. ► Demonstration that recombinant PnTx3-4 shows biological activity similar to the native peptide using two different assays. ► Suggestion that PnTx3-4 adopts a knottin scaffold based on primary sequence analysis. ► Circular dichroism analysis showing that the peptide is composed of about 53% turns/unordered, 31% α-helix and 16% β-strand.