Dai Hui, Yin Shijin, Li Tian, Cao Zhijian, Ji Yonghua, Wu Yingliang, Li Wenxin
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Protein Expr Purif. 2012 Apr;82(2):325-31. doi: 10.1016/j.pep.2012.02.001. Epub 2012 Feb 10.
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.
长链且富含半胱氨酸的蝎毒素对不同的电压门控钠通道亚型表现出多种药理学特性。然而，由于难以获得合成或重组肽，毒素结构-功能关系的探索进展缓慢。我们现在报告，我们已经为新型蝎毒素BmαTX14建立了一种有效的表达和纯化方法。BmαTX14在大肠杆菌中作为包涵体过量表达。通过复性程序成功地将不溶性沉淀转化为活性肽。通过反相高效液相色谱一步纯化足以产生色谱纯的肽。从1升LB培养基中重组毒素的产量达到4毫克。药理学数据进一步表明，BmαTX14选择性抑制mNa(v)1.4的快速失活（EC(50)=82.3±15.7 nM），而不是rNa(v)1.2的快速失活（EC(50)>30μM），这表明BmαTX14是一种新的α样毒素。这项工作使得能够对BmαTX14和类似毒素进行进一步的结构、功能和药理学研究。
