title: Design, synthesis, and functional expression of a gene for charybdotoxin, a peptide blocker of K+ channels.
abstract: A gene encoding charybdotoxin (CTX), a K+ channel blocker from scorpion venom, was designed, synthesized, and expressed as a cleavable fusion protein in Escherichia coli. A sequence-specific protease, factor Xa, was used to cleave the fusion protein and thus release the toxin peptide. The recombinant toxin was purified, oxidized to form disulfide bonds, and treated to form N-terminal pyroglutamate. Recombinant CTX is identical to the native venom CTX with respect to high-performance liquid chromatography mobility, amino acid composition, and N-terminal modification. With single Ca2(+)-activated K+ channels as an assay system, recombinant CTX shows blocking and dissociation kinetics identical to the native venom toxin. The synthetic gene and high-level expression of functionally active CTX make it possible to study the fundamental mechanism of the toxin-ion channel interaction.
authors.affiliation: Howard Hughes Medical Institute, Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254.
authors.forename: C S
authors.initials: CS
authors.lastname: Park
authors.forename: S F
authors.initials: SF
authors.lastname: Hausdorff
authors.forename: C
authors.initials: C
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018999:Charybdotoxin; D004926:Escherichia coli / Q000235:genetics*; D015951:Factor Xa / Q000378:metabolism; D005813:Genes, Synthetic; D007700:Kinetics; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000187:drug effects* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D012604:Scorpion Venoms / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D012605:Scorpions
chemical_list: D008051:Lipid Bilayers; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; D018999:Charybdotoxin; D015951:Factor Xa