title: Recombinant expression, purification, and characterization of scorpion toxin BmαTX14.
abstract: Long-chain and cysteine-rich scorpion toxins exhibit various pharmacological profiles for different voltage-gated sodium channel subtypes. However, the exploration of toxin structure-function relationships has progressed slowly due to the difficulty of obtaining synthetic or recombinant peptides. We now report that we have established an effective expression and purification approach for the novel scorpion toxin BmαTX14. BmαTX14 was over-expressed as inclusion bodies in Escherichia coli. The insoluble pellet was successfully transformed into active peptide by using a refolding procedure. One-step purification by reverse-phase HPLC was sufficient to generate chromatographically pure peptide. The yield of recombinant toxin reached 4mg from 1L LB medium. The pharmacological data further showed that BmαTX14 selectively inhibited the fast inactivation of mNa(v)1.4 (EC(50)=82.3±15.7nM) rather than that of rNa(v)1.2 (EC(50)>30μM), which indicates that BmαTX14 is a new α-like toxin. This work enables further structural, functional, and pharmacological studies of BmαTX14 and similar toxins.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
authors.forename: Hui
authors.initials: H
authors.lastname: Dai
authors.forename: Shijin
authors.initials: S
authors.lastname: Yin
authors.forename: Tian
authors.initials: T
authors.lastname: Li
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Cao
authors.forename: Yonghua
authors.initials: Y
authors.lastname: Ji
authors.forename: Yingliang
authors.initials: Y
authors.lastname: Wu
authors.forename: Wenxin
authors.initials: W
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D019476:Insect Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D012604:Scorpion Venoms / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D026941:Sodium Channel Blockers / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; D026941:Sodium Channel Blockers; C518690:alpha-neurotoxin TX14, Buthus martensii