Recombinant expression, purification, and characterization of scorpion toxin BmαTX14
Recombinant expression, purification, and characterization of scorpion toxin BmαTX14
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DOI：
10.1016/j.pep.2012.02.001
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发表时间：
2012-04-01
影响因子：
1.6
通讯作者：
Li, Wenxin
中科院分区：
文献类型：
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作者：
Dai, Hui;Yin, Shijin;Li, Wenxin
长链和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不同的药理学特征。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，我们已经建立了一个有效的表达和纯化方法的新型蝎毒素Bm α TX 14。Bm α TX 14在大肠杆菌中以包涵体形式过量表达。不溶性沉淀通过使用复性程序成功地转化为活性肽。通过反相HPLC的一步纯化足以产生色谱纯的肽。每升LB培养基的重组毒素产量可达4 mg。药理学数据进一步表明，Bm α TX 14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> 30 μ M），这表明Bm α TX 14是一种新的α样毒素。这项工作使Bm alpha TX 14和类似毒素的结构，功能和药理学研究成为可能。(C)2012 Elsevier Inc. All rights reserved.
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 alpha TX14. Bm alpha 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 alpha TX14 selectively inhibited the fast inactivation of mNa(v)1.4 (EC50 = 82.3 +/- 15.7 nM) rather than that of rNa(v)1.2 (EC50 > 30 mu M), which indicates that Bm alpha TX14 is a new alpha-like toxin. This work enables further structural, functional, and pharmacological studies of Bm alpha TX14 and similar toxins. (C) 2012 Elsevier Inc. All rights reserved.