Improving extraction and post-purification concentration of membrane proteins
Improving extraction and post-purification concentration of membrane proteins
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DOI：
10.1039/c7an01470h
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发表时间：
2018-03-21
期刊：
影响因子：
4.2
通讯作者：
Kumar, Manish
中科院分区：
文献类型：
--
作者：
Feroz, Hasin;Kwon, HyeYoung;Kumar, Manish
膜蛋白（MPs）尽管是制药行业至关重要的药物靶点，但由于难以获得高产量的功能蛋白，因此很难对其进行研究。目前大多数的萃取方法都是使用特殊的非离子洗涤剂来溶解和稳定MPs，并通过超滤（UF）来浓缩MPs。然而，在超滤步骤中保留了许多洗涤剂，这会破坏MPs的稳定性和/或干扰它们的表征。在这里，我们研究了洗涤剂选择对生物医学相关MPs、光驱动钠和氯转运体、KR2和盐紫红质（pHR）的提取和基于uf的浓度的影响，这些蛋白也是更复杂的哺乳动物视紫红质的模型蛋白。我们还设计了一种平底离心过滤器，可以通过促进浓度极化（CP）来浓缩MPs，同时增强对游离洗涤剂的去除。我们使用四种常用的MP洗涤剂，辛烷基-D麦芽糖苷（OM），癸基-D麦芽糖苷（DM），十二烷基-D麦芽糖苷（DDM）和辛烷基-D葡萄糖苷（OG），在一系列洗涤剂和盐浓度下测试了这种新型过滤器的性能。由于CP的高度，在dm溶解的pHR系统中，平底过滤器的洗涤剂通过率显著提高，洗涤剂的筛分率提高了2倍。我们观察到，与较短的8碳洗涤剂OM相比，在较长的12碳烷基链洗涤剂DDM中，KR2的提取率提高了5倍。假设提取的蛋白质完全结合和洗脱，基于ddm的KR2提取可能比传统方法纯化产量提高7倍，传统方法每升细胞培养产量约为1 mg MP。然而，像DDM这样的长链洗涤剂形成较大的胶束，很难被UF去除。因此，在选择一种既能有效提取MP又能在随后的UF中更容易去除的洗涤剂之间存在权衡。洗涤剂胶束的提取效率和基于uf的分离为涉及洗涤剂介导的分离/提取的其他应用提供了见解。
Membrane proteins (MPs), despite being critically important drug targets for the pharmaceutical industry, are difficult to study due to challenges in obtaining high yields of functional protein. Most current extraction efforts use specialized non-ionic detergents to solubilize and stabilize MPs, with MPs being concentrated by ultrafiltration (UF). However, many detergents are retained during the UF step, which can destabilize MPs and/or interfere with their characterization. Here, we studied the influence of detergent selection on the extraction and UF-based concentration of biomedically-relevant MPs, the light-driven sodium and chloride transporters, KR2 and halorhodopsin (pHR) which are also model proteins for more complex mammalian rhodopsins. We also designed a flat-bottomed centrifugal filter that can concentrate MPs with enhanced removal of free detergents by promoting concentration polarization (CP). We tested the performance of this new filter using four commonly employed MP detergents, octyl--D maltoside (OM), decyl--D maltoside (DM), dodecyl--D maltoside (DDM) and octyl--D glucoside (OG), over a range of detergent and salt concentrations. Detergent passage is significantly higher for the flat-bottomed filter achieving up to 2-fold greater sieving of detergent in DM-solubilized pHR system due to the high degree of CP. We observe more efficient, up to 5-fold higher extraction of KR2 in the presence of a longer 12-carbon alkyl chain detergent, DDM compared to a shorter 8-carbon detergent, OM. Assuming complete binding and elution of the extracted protein, DDM-based extraction of KR2 could lead to a potential 7-fold improvement in purification yields compared to conventional methods which yield approximate to 1 mg MP per liter of cell culture. However, the longer chain detergents like DDM form larger micelles that are difficult to remove by UF. Thus, there exists a trade-off between choosing a detergent that will enable efficient extraction of MP while showing easier removal during subsequent UF. The extraction efficiency and UF-based separation of detergent micelles provide insights for other applications involving detergent-mediated separation/extraction.