Overexpression, purification, and pharmacological activity of a biosynthetically derived conopeptide

Abstract

A high yielding fusion protein system based on the protein cytochrome b₅ has been used for the production of novel 13-residue acyclic conopeptide. This peptide, Mo1659, can be liberated from the carrier protein using CNBr cleavage and subsequent purification using RP-HPLC methods. The yield of isotopically enriched peptides is high, ranging from 3 to 4 mg of purified peptide from a 500 ml culture, indicating that this system can be widely used for peptide production. Biosynthetic Mo1659 is active on non-inactivating K⁺ channel much like the natural Mo1659, despite the absence of C-terminal amidation. Heteronuclear NMR studies show that the peptide exists in a conformational equilibrium involving proline-10. To our knowledge this is the first report of the production of an isotopically ¹⁵N/¹³C-enriched conopeptide.