Automated and manual solid phase peptide synthesis techniques were combined with chemical ligation to produce a 37-residue peptide toxin derivative of iberiotoxin which contained: (i) substitution of Val 16 to Ala, to facilitate kinetic feasibility of native chemical ligation, and; (ii) substitution of Asp 19 to orthogonally protected Cys-4-MeOBzl for chemical conjugate derivatization following peptide folding and oxidation. This peptide ligation approach increased synthetic yields approximately 12-fold compared to standard linear peptide synthesis. In a functional inhibition assay, the ligated scorpion toxin derivative, iberiotoxin V16A/D19-Cys-4-MeOBzl, exhibited 'native-like' affinity (K d = 1.9 nM) and specificity towards the BK Ca 2+ -activated K + Channel (K Ca 1.1). This was characterized by the rapid association and slow dissociation rates (k on = 4.59 × 10 5 M −1 s −1 ; k off = 8.65 × 10 −4 s −1 ) as determined by inhibition of macroscopic whole-cell currents of cloned human K Ca 1.1 channel. These results illustrate the successful application of peptide chemical ligation to improve yield of cysteine-rich peptide toxins over traditional solid phase peptide synthesis. Native chemical ligation is a promising method for improving production of biologically active disulfide containing peptide toxins, which have diverse applications in studies of ion-channel function.

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