Recombinant expression of margatoxin and agitoxin-2 in Pichia pastoris: an efficient method for production of KV1.3 channel blockers.
Recombinant expression of margatoxin and agitoxin-2 in Pichia pastoris: an efficient method for production of KV1.3 channel blockers.
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DOI：
10.1371/journal.pone.0052965
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
King GF
中科院分区：
文献类型：
--
作者：
Anangi R;Koshy S;Huq R;Beeton C;Chuang WJ;King GF
Kv1.3电压门控钾通道调节人效应记忆T细胞中的膜电位和钙信号传导，这些细胞是自身免疫性疾病如多发性硬化症、1型糖尿病和类风湿性关节炎的关键介质。因此，亚型特异性Kv1.3阻断剂具有治疗自身免疫性疾病的潜力。已经从蝎毒中鉴定了几种Kv1.3通道阻断剂，它们都具有由3-4个分子内二硫键稳定的α/β支架。化学合成通常用于生产这些富含二硫键的肽，但这种方法耗时，并且对于生产突变体、融合蛋白、荧光标记的毒素或用于NMR研究的同位素标记的肽而言不具有成本效益。Kv1.3阻断剂在E.大肠杆菌通常需要肽的氧化重折叠以形成它们的天然二硫键结构。另一种避免重折叠的方法是在大肠杆菌的周质中表达肽。但这通常产生低产量。因此，我们开发了一个有效的巴斯德毕赤酵母表达系统，用于生产Kv1.3阻断剂，使用玛格毒素（MgTx）和agitoxin-2（AgTx 2）作为原型的例子。毕赤酵母系统能够以高产率（12-18 mg/L）获得这些毒素。NMR实验显示重组毒素采用其天然折叠而不需要再折叠，并且电生理学记录证明它们在阻断KV1.3方面几乎与天然毒素等效（重组AgTx 2和MgTx的IC 50值分别为201±39 pM和97±3 pM）。此外，这两种重组毒素抑制T淋巴细胞增殖。其中关键药效团残基K28突变为丙氨酸的MgTx突变体在阻断KV1.3方面是无效的，并且它未能抑制T淋巴细胞增殖。因此，本文所述的方法提供了一种生产毒素突变体的有效方法，以期工程化具有治疗潜力的Kv1.3阻断剂。
The Kv1.3 voltage-gated potassium channel regulates membrane potential and calcium signaling in human effector memory T cells that are key mediators of autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. Thus, subtype-specific Kv1.3 blockers have potential for treatment of autoimmune diseases. Several Kv1.3 channel blockers have been characterized from scorpion venom, all of which have an α/β scaffold stabilized by 3–4 intramolecular disulfide bridges. Chemical synthesis is commonly used for producing these disulfide-rich peptides but this approach is time consuming and not cost effective for production of mutants, fusion proteins, fluorescently tagged toxins, or isotopically labelled peptides for NMR studies. Recombinant production of Kv1.3 blockers in the cytoplasm of E. coli generally necessitates oxidative refolding of the peptides in order to form their native disulfide architecture. An alternative approach that avoids the need for refolding is expression of peptides in the periplasm of E. coli but this often produces low yields. Thus, we developed an efficient Pichia pastoris expression system for production of Kv1.3 blockers using margatoxin (MgTx) and agitoxin-2 (AgTx2) as prototypic examples. The Pichia system enabled these toxins to be obtained in high yield (12–18 mg/L). NMR experiments revealed that the recombinant toxins adopt their native fold without the need for refolding, and electrophysiological recordings demonstrated that they are almost equipotent with the native toxins in blocking KV1.3 (IC50 values of 201±39 pM and 97±3 pM for recombinant AgTx2 and MgTx, respectively). Furthermore, both recombinant toxins inhibited T-lymphocyte proliferation. A MgTx mutant in which the key pharmacophore residue K28 was mutated to alanine was ineffective at blocking KV1.3 and it failed to inhibit T-lymphocyte proliferation. Thus, the approach described here provides an efficient method of producing toxin mutants with a view to engineering Kv1.3 blockers with therapeutic potential.
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