Isolation, cloning, expression and purification of alpha-conotoxin PnIB from the cone snail Conus pennaceus of the Persian Gulf

Background and Aim: Conotoxins are small peptide neurotoxins from cone snails. These toxins have a stable structure that is rich in disulfide bond and are able to bind to ion channels, neurotransmitter receptors and transporters in nerve cells. These toxins are used to target and study diseases of the nervous system such as neuropathic pain. The aim of this study was isolation, cloning, expression and purification of α-conotoxin PnIB (conotoxin PnIB) from the cone snail Conus pennaceus of the Persian Gulf.Methods: In this study, specimens of C. pennaceus were collected from Qeshm Island in the Persian Gulf and DNA extraction was performed from its tissue. Then, using designed specific primers, the conotoxin PnIB gene fragment was amplified. The gene fragment was cloned into the expression vector pET۳۲b(+). After DNA sequencing, a bioinformatic study was performed to compare its nucleotide and peptide sequences with similar reported sequences. The recombinant vector was transformed and expressed in the expression host of Escherichia coli BL۲۱(DE۳). Finally, the fusion protein thioredoxin–conotoxin PnIB was purified by Ni-NTA affinity chromatography.Results: In this study, the gene sequence encoding conotoxin PnIB with ۴۸ nucleotides long was isolated from the C. pennaceus. This conotoxin has ۱۶ amino acids, two disulfide bonds and an alpha-helix structure that is classified as alpha-conotoxins. The conotoxin PnIB gene was cloned into the prokaryotic expression vector pET۳۲b(+) and its expression was observed as a fusion with thioredoxin in the soluble phase. After purification of the fusion protein, the thioredoxin was separated by enterokinase and the concentration of purified protein was approximately ۲۳۲ mg / L.Conclusion: These results show that the production of small conotoxins containing disulfide bonds as a recombinant protein could lead to significant peptide production for future studies.

کلیدواژه ها:

Cone snail ، Conus pennaceus ، Alpha-conotoxin PnIB.