Conkunitzin-S1 from the cone snail Conus striatus is the first member of a new neurotoxin family with a canonical Kunitz domain fold. Conk-SI is 60 amino acids long and lacks one of the three conserved disulfide bonds typically found in Kunitz domain modules. It binds specifically to voltage activated potassium channels of the Shaker family. The peptide was expressed in insoluble form in fusion with an N-terminal intein. Refolding in the presence of glutathione followed by pH shift-induced cleavage of the fusion protein resulted in a functional toxin as demonstrated by voltage-clamp measurements. (c) 2006 Elsevier Inc. All rights reserved.