Factors influencing the detergent-free membrane protein isolation using synthetic nanodisc-forming polymers.
Factors influencing the detergent-free membrane protein isolation using synthetic nanodisc-forming polymers.
复制标题
影响使用合成纳米盘形成聚合物进行无去污剂膜蛋白分离的因素。
DOI：
10.1101/2023.05.12.540572
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Ramamoorthy,Ayyalusamy
中科院分区：
文献类型：
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作者：
Krishnarjuna,Bankala;Sharma,Gaurav;Ravula,Thirupathi;Ramamoorthy,Ayyalusamy
使用合成聚合物进行膜蛋白的无去污剂分离正在成为膜蛋白功能和结构研究的理想方法。由于许多膜蛋白的表达水平较低，并且功能化重组膜蛋白的高产率对于体外研究至关重要，因此优化给定聚合物的实验条件以有效溶解目标膜/蛋白质至关重要。影响膜溶解和随后目标膜蛋白分离的因素包括聚合物浓度、聚合物电荷、温度、pH 和二价金属离子浓度。因此，了解不同类型聚合物溶解细胞膜的功效非常重要。在这项研究中，我们评估了基于菊粉的非离子聚合物在溶解大肠杆菌方面的功效。在各种溶解条件下富含细胞色素 P450 还原酶 (CPR) 的大鼠黄素单核苷酸结合域 (FBD) 和兔细胞色素 -b5 (Cyt-b5) 的共膜。我们的结果表明，1:1 (w/w) 的膜：聚合物比例、低温、高 pH 值和亚毫摩尔浓度的金属离子有利于大肠杆菌的溶解。富含 FBD 或 Cyt-b5 的共膜。相反，过量二价金属离子的存在会影响聚合物溶解样品中的最终蛋白质水平。我们相信，这项研究的结果为评估和规划非离子聚合物在膜蛋白研究中的使用提供了知识。
The detergent-free isolation of membrane proteins using synthetic polymers is becoming the desired approach for functional and structural studies of membrane proteins. Since the expression levels for many membrane proteins are low and a high yield of functionalized reconstituted membrane proteins is essential forin vitrostudies, it is crucial to optimize the experimental conditions for a given polymer to solubilize target membranes/proteins effectively. The factors that affect membrane solubilization and subsequently the isolation of a target membrane protein include polymer concentration, polymer charge, temperature, pH, and concentration of divalent metal ions. Therefore, it is important to have knowledge about the efficacy of different types of polymers in solubilizing cell membranes. In this study, we evaluate the efficacy of inulin-based non-ionic polymers in solubilizingE. colimembranes enriched with rat flavin mononucleotide binding-domain (FBD) of cytochrome-P450-reductase (CPR) and rabbit cytochrome-b5 (Cyt-b5) under various solubilization conditions. Our results show that a 1:1 (w/w) membrane:polymer ratio, low temperature, high pH and sub-millimolar concentration of metal ions favor the solubilization ofE. colimembranes enriched with FBD or Cyt-b5. Conversely, the presence of excess divalent metal ions affected the final protein levels in the polymer-solubilized samples. We believe that the results from this study provide knowledge to assess and plan the use of non-ionic polymers in membrane protein studies.