Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.
Biochemistry (Mosc). 2009 Oct;74(10):1142-9. doi: 10.1134/s0006297909100101.
The gene for the "weak" toxin of Naja kaouthia venom was expressed in Escherichia coli. "Weak" toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of "weak" toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia "weak" toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain "weak" toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of (125)I-labeled alpha-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (alpha1(2)beta1gammadelta) showed the presence of biological activity of the recombinant "weak" toxin close to the activity of the natural toxin (IC(50) = 4.3 +/- 0.3 and 3.0 +/- 0.5 microM, respectively). The interaction of the recombinant toxin with alpha7 type human neuronal acetylcholine receptor transfected in the GH(4)C(1) cell line also showed the presence of activity close to that of the natural toxin (IC(50) 31 +/- 5.0 and 14.8 +/- 1.3 microM, respectively). The developed bacterial system for production of N. kaouthia venom "weak" toxin was used to obtain (15)N-labeled analog of the neurotoxin.
眼镜蛇科纳加毒蛇的“弱”毒素基因在大肠杆菌中表达。“弱”毒素是烟碱型乙酰胆碱受体的特异性抑制剂，但类似神经毒素与受体相互作用的机制仍不清楚。先前为来自眼镜蛇 Naja oxiana 毒液的神经毒素 II 开发的系统被用于测试从 N. kaouthia 毒液中细菌生产“弱”毒素。设计了用于在硫氧还蛋白和 STII 前导肽存在下以融合多肽链的形式在细胞质中产生 N. kaouthia“弱”毒素的构建体，并用于分泌。然而，只有在细胞质包涵体中才能以毫克量获得“弱”毒素。测试了不同的复性方法，并研究了在复性过程中优化目标蛋白产率的条件。所得蛋白通过质谱法和 CD 及 NMR 光谱法进行了表征。用竞争抑制实验研究了（125）I 标记的α-银环蛇毒素与含有肌肉型烟碱型乙酰胆碱受体（α1（2）β1γδε）的加利福尼亚电鳐电器官膜结合的实验，结果表明重组“弱”毒素具有与天然毒素相近的生物活性（IC50 分别为 4.3 ± 0.3 和 3.0 ± 0.5 μM）。在 GH（4）C（1）细胞系中转染的重组毒素与人神经元乙酰胆碱受体α7 型的相互作用也表明其活性与天然毒素相近（IC50 分别为 31 ± 5.0 和 14.8 ± 1.3 μM）。开发的用于生产 N. kaouthia 毒液“弱”毒素的细菌系统用于获得神经毒素的（15）N 标记类似物。
