Cloning and functional expression of dendrotoxin K from black mamba, a potassium channel blocker

Abstract

Mamba dendrotoxins, 7K M(r) polypeptides with three disulfide bonds, selectively inhibit certain fast-activating, voltage-sensitive neuronal K+ 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+ channels in bovine synaptic membranes. This successful expression of refolded active toxin, in adequate yield, makes possible for the first time the preparation of mutants with specificity tailored for each K+ channel subtype, based both on the recently derived three-dimensional structure of alpha-dendrotoxin and the identified binding site on cloned K+ channels.

SdPI, The First Functionally Characterized Kunitz-Type Trypsin Inhibitor from Scorpion Venom

“…Multiple sequence alignments showed that mature SdPI shares homology with typical Kunitz-type venom peptides, including HWTX-XI from spider [7], kalicludine-1 from sea anemone [6], DTX-K from snake [8], and conkunitizin-S1 from cone snail [17]. The most homologous sequence HWTX-XI shows 52.7% identity to SdPI.…”

“…Currently reported Kunitz-type venom peptides can be classified into two families based on different cysteine frameworks. One family retains the typical Kuntiz-type architecture, with three highly conserved disulfide bridges, exemplified by HWTX-XI from spider, DTX-K from snake, and kalicludines from sea anemone [6], [7], [8]. The other family has only four cysteine residues, which results in the apparent ‘loss’ of a conserved disulfide bridge, represented by conkunitzin-S1 from cone snail [9].…”

Functional Expression of Spider Neurotoxic Peptide Huwentoxin-I in E. coli

“…Compared with the cytoplasm, the periplasm of E. coli cells provides a relatively non-reducing environment that allows disulfide bridges to form [28]. It was reported that several peptide toxins, including Dendrotoxin K and Huwentoxin-XI, were functionally expressed in the periplasm of E. coli with the formation of disulfide bridges found in their natural forms [29], [30].…”

Hg1, Novel Peptide Inhibitor Specific for Kv1.3 Channels from First Scorpion Kunitz-type Potassium Channel Toxin Family

“…1). Hg1 and BmKTT-3 belong to the first group, which adopted classical disulfide pairings similar to HWTX-XI toxin from spider (20), dendrotoxin K from snake (32), and APEKTx1 from sea anemone (23). LmKTT-1a, LmKTT-1b, LmKTT-1c, and BmKTT-1 belong to the second group, which adopted a unique cystine framework that we described in our previous work (26).…”