title: Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom.
abstract: A novel peptidyl inhibitor of voltage-gated K+ channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called 'alpha-KTx1.1'). PBTx3 is the tenth member (called 'alpha-KTx1.10') of subfamily 1 of K+ channel-blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels. The dissociation constants (Kd) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 microm, 547 nm and 492 nm. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor Xa (fXa) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The three-dimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel beta sheet and a single alpha-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other alpha-KTx scorpion toxins.
authors.affiliation: Laboratory of Toxicology, University of Leuven, Leuven, Belgium.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Huys
authors.forename: Karin
authors.initials: K
authors.lastname: Dyason
authors.forename: Etienne
authors.initials: E
authors.lastname: Waelkens
authors.forename: Fons
authors.initials: F
authors.lastname: Verdonck
authors.forename: Johann
authors.initials: J
authors.lastname: van Zyl
authors.forename: Johan
authors.initials: J
authors.lastname: du Plessis
authors.forename: Gert J
authors.initials: GJ
authors.lastname: Müller
authors.forename: Jurg
authors.initials: J
authors.lastname: van der Walt
authors.forename: Elke
authors.initials: E
authors.lastname: Clynen
authors.forename: Liliane
authors.initials: L
authors.lastname: Schoofs
authors.forename: Jan
authors.initials: J
authors.lastname: Tytgat
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D015640:Ion Channel Gating; D008958:Models, Molecular; D008969:Molecular Sequence Data; D026902:Potassium Channel Blockers; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012604:Scorpion Venoms / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D012605:Scorpions; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers; D026902:Potassium Channel Blockers; D011994:Recombinant Proteins; D012604:Scorpion Venoms; C454815:parabutoxin 3 protein, Parabuthus transvaalicus