Conotoxin PrIIIE is a 22-amino acid peptide containing three disulfide bonds isolated from the venom of Con us parius Reeve. It is a non-competitive antagonist of the mammalian muscle nicotinic acetylcholine receptor (nAChR). We fused the PrIllE 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 PrIllE from cleaved SUMO using cation exchange chromatography. The yield of peptide was 1.5 mg/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. (C) 2011 Elsevier Inc. All rights reserved.