Expression of a biologically-active conotoxin PrIIIE in Escherichia coli

► Conotoxins are valuable tools for determining structure–function relationships in ion channels. ► Recombinant expression of conotoxins can produce many mutant toxins. ► We produced a fully functional recombinant PrIIIe conotoxin. Conotoxin PrIIIE is a 22-amino acid peptide containing three disulfide bonds isolated from the venom of Conus parius Reeve. It is a non-competitive antagonist of the mammalian muscle nicotinic acetylcholine receptor (nAChR). We fused the PrIIIE to small ubiquitin-like modifier (SUMO) and expressed the fusion protein in an Escherichia coli strain with an oxidizing cytoplasm. We purified the fusion protein by immobilized metal affinity chromatography and further purified PrIIIE from cleaved SUMO using cation exchange chromatography. The yield of peptide was 1.5mg/L of culture. The recombinant peptide is functional, as demonstrated by two-electrode voltage clamp experiments. This system may prove valuable for future structure–function studies.