Cloning and functional expression of dendrotoxin K from black mamba, a K super(+) channel blocker

Mamba dendrotoxins, 7K M sub(r) polypeptides with three disulfide bonds, selectively inhibit certain fast-activating, voltage-sensitive neuronal K super(+) channels and have been instrumental in their identification, localization, and purification. However, derivatives with more refined specificity are essential to define the structural and functional properties of the multiple subtypes known to reside in the nervous system. Hence, utilizing a constructed cDNA library from the venom glands of the black mamba (Dendroaspis polylepis)), the gene encoding dendrotoxin K was isolated, amplified, and expressed as a maltose-binding fusion protein in the periplasmic space of Escherichia coli . After cleavage of the chaperone from the affinity-purified product, a recombinant protein was isolated and shown to be identical to native dendrotoxin K in its N-terminal sequence, chromatographic behavior, convulsive-inducing activity, and binding to voltage-activated K super(+) channels in bovine synaptic membranes.