Cloning and functional expression of dendrotoxin K from black mamba, a K+ channel blocker.
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.
Smith
L A
LA
Department of Immunology and Molecular Biology, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702-5011.
Lafaye
P J
PJ
LaPenotiere
H F
HF
Spain
T
T
Dolly
J O
JO
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism; D003001:Cloning, Molecular; D004546:Elapid Venoms / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005796:Genes; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000187:drug effects*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012911:Snakes / Q000235:genetics*; D013570:Synaptic Membranes / Q000378:metabolism*
D004546:Elapid Venoms; D009498:Neurotoxins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D015221:Potassium Channels; D011994:Recombinant Proteins; C081506:dendrotoxin K; C020273:dendrotoxin