Recombinant expression, purification, and characterization of scorpion toxin BmαTX14

• Published in: Protein expression and purification 2012-04, Vol.82 (2), p.325-331
• Main Authors: Dai, Hui, Yin, Shijin, Li, Tian, Cao, Zhijian, Ji, Yonghua, Wu, Yingliang, Li, Wenxin
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; BmαTX14; Chromatography, Affinity; Dose-Response Relationship, Drug; Expression and purification; HEK293 Cells; Humans; Insect Proteins - biosynthesis; Insect Proteins - isolation & purification; Insect Proteins - pharmacology; Membrane Potentials - drug effects; Mice; Modulator; Molecular Sequence Data; Rats; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - pharmacology; Refolding in vitro; Scorpion Venoms - biosynthesis; Scorpion Venoms - isolation & purification; Scorpion Venoms - pharmacology; Scorpion α-like toxin; Sodium channel; Sodium Channel Blockers - isolation & purification; Sodium Channel Blockers - pharmacology
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2012.02.001

► Toxin rBmαTX14 was produced by the recombinant expression in Escherichia coli. ► BmαTX14’s structure is stable with or without His6-tag. ► BmαTX14 selectively inhibits the fast inactivation of mNav1.4 channel. ► The activity of BmαTX14 inhibiting channel inactivation is less affected by His6-tag. 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 mNav1.4 (EC50=82.3±15.7nM) rather than that of rNav1.2 (EC50>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.