Membrane solubilization by detergents is a critical step for successful membrane protein purification. Alkyl maltoside detergents such as DDM and DM are very expensive and are commonly used to produce most of the high-quality proteins in stable and functional form. Recently, dual-detergent strategy using inexpensive detergents for membrane solubilization step has been shown to be highly effective in purifying different classes of membrane proteins in a cost-effective manner. In this work, we have monitored the effectiveness of ‘dual-detergent strategy’ towards successful purification of the isolated voltage sensing domain (VSD) of KvAP and the inward rectifying K⁺ channel, KirBac1.1. We demonstrate that the inexpensive detergent Triton X-100 extracts the activated conformation of the KvAP-VSD well without compromising the structural integrity of the sensor, and also retains its proper structural dynamics. Importantly, the cost associated with solubilizing the KvAP sensor can be reduced by ∼2000 fold. To the best of our knowledge, our results constitute the first report characterizing the purification of KvAP voltage sensor using an inexpensive detergent. However, the dual-detergent strategy using Triton X-100 for membrane solubilization is not effective for the purification of inward rectifying K⁺ channel, KirBac1.1 even in presence of high salt concentration during solubilization. We propose that the dual-detergent strategy will be useful for extracting stable and functional proteins that are both DDM- and DM-extractable, but will be ineffective if the protein is only DM-extractable. The relevance of the effectiveness of dual-detergent strategy with respect to the hydrophobic thickness of proteins is discussed.

去污剂对膜的溶解是成功纯化膜蛋白的关键步骤。DDM 和 DM 等烷基麦芽糖苷去污剂非常昂贵，通常用于生产大多数稳定和功能性形式的高质量蛋白质。最近，使用廉价去污剂进行膜溶解步骤的双去污剂策略已被证明在以具有成本效益的方式纯化不同类别的膜蛋白方面非常有效。在这项工作中，我们监测了“双去污剂策略”对成功纯化 KvAP 的隔离电压传感域 (VSD) 和内向整流 K ^{+的效果。}通道，KirBac1.1。我们证明了廉价的洗涤剂 Triton X-100 可以很好地提取 KvAP-VSD 的活化构象，而不会损害传感器的结构完整性，并且还保留了其适当的结构动力学。重要的是，与溶解 KvAP 传感器相关的成本可以降低约 2000 倍。据我们所知，我们的结果构成了第一份描述使用廉价清洁剂纯化 KvAP 电压传感器的报告。然而，使用Triton X-100进行膜增溶的双重去污剂策略对内向精馏K ^{+的纯化效果不佳}通道，KirBac1.1 即使在溶解过程中存在高盐浓度。我们建议双去污剂策略可用于提取稳定的功能性蛋白质，这些蛋白质既可被 DDM 提取，又可被 DM 提取，但如果蛋白质仅可被 DM 提取，则将无效。讨论了双洗涤剂策略的有效性与蛋白质疏水厚度的相关性。