Recombinant expression, purification, and characterization of scorpion toxin Bm伪TX14

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• 作者：Hui Dai^a ; ¹ ; Shijin Yin^b ; ¹ ; Tian Li^a ; Zhijian Cao^a ; Yonghua Ji^c ; Yingliang Wu^a ; ^{ylwu@whu.edu.cn} ; Wenxin Li^a ; ^{liwxlab@whu.edu.cn}
• 关键词：Expression and purification ; Scorpion 伪-like toxin ; Bm伪TX14 ; Sodium channel ; Refolding in vitro ; Modulator
• 刊名：Protein Expression and Purification
• 出版年：2012
• 期刊代码：184_10465928
• 类别：bio
• 出版时间：April, 2012
• 卷：82
• 期：2
• 页码：325-331
• 文件大小：754 K

摘要

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 Bm伪TX14. Bm伪TX14 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 4 mg from 1 L LB medium. The pharmacological data further showed that Bm伪TX14 selectively inhibited the fast inactivation of mNa_v1.4 (EC₅₀ = 82.3 卤 15.7 nM) rather than that of rNa_v1.2 (EC₅₀ > 30 渭M), which indicates that Bm伪TX14 is a new 伪-like toxin. This work enables further structural, functional, and pharmacological studies of Bm伪TX14 and similar toxins.