Pollock Naomi L, McDevitt Christopher A, Collins Richard, Niesten Petronella H M, Prince Stephen, Kerr Ian D, Ford Robert C, Callaghan Richard
Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK.
Biochim Biophys Acta. 2014 Jan;1838(1 Pt B):134-47. doi: 10.1016/j.bbamem.2013.09.001. Epub 2013 Sep 12.
ATP Binding Cassette (ABC) transporters play prominent roles in numerous cellular processes and many have been implicated in human diseases. Unfortunately, detailed mechanistic information on the majority of ABC transporters has not yet been elucidated. The slow rate of progress of molecular and high resolution structural studies may be attributed to the difficulty in the investigation of integral membrane proteins. These difficulties include the expression of functional, non-aggregated protein in heterologous systems. Furthermore, the extraction of membrane proteins from source material remains a major bottle-neck in the process since there are relatively few guidelines for selection of an appropriate detergent to achieve optimal extraction. Whilst affinity tag strategies have simplified the purification of membrane proteins; many challenges remain. For example, the chromatographic process and associated steps can rapidly lead to functional inactivation, random aggregation, or even precipitation of the target protein. Furthermore, optimisation of high yield and purity, does not guarantee successful structure determination. Based on this series of potential issues, any investigation into structure-function of membrane proteins requires a systematic evaluation of preparation quality. In particular, the evaluation should focus on function, homogeneity and mono-dispersity. The present investigation provides a detailed assessment of the quality of purified ATP Binding Cassette (ABC) transporters; namely ABCB1 (P-gp) and ABCA4 (ABCR). A number of suggestions are provided to facilitate the production of functional, homogeneous and mono-disperse preparations using the insect cell expression system. Finally, the ABCA4 samples have been used to provide structural insights into this essential photo-receptor cell protein.
ATP结合盒（ABC）转运蛋白在众多细胞过程中发挥着重要作用，许多已被证实与人类疾病有关。不幸的是，大多数ABC转运蛋白的详细机制信息尚未阐明。分子和高分辨率结构研究进展缓慢可能归因于对整合膜蛋白进行研究存在困难。这些困难包括在异源系统中表达功能性、非聚集性蛋白。此外，从原材料中提取膜蛋白仍然是该过程中的一个主要瓶颈，因为选择合适的去污剂以实现最佳提取的指导原则相对较少。虽然亲和标签策略简化了膜蛋白的纯化；但仍存在许多挑战。例如，色谱过程及相关步骤可能会迅速导致目标蛋白功能失活、随机聚集甚至沉淀。此外，高产率和高纯度的优化并不能保证成功确定结构。基于这一系列潜在问题，对膜蛋白结构功能的任何研究都需要对制备质量进行系统评估。特别是，评估应侧重于功能、均匀性和单分散性。本研究对纯化的ATP结合盒（ABC）转运蛋白，即ABCB1（P-糖蛋白）和ABCA4（ABCR）的质量进行了详细评估。提供了一些建议，以促进使用昆虫细胞表达系统生产功能性、均匀性和单分散性的制剂。最后，已使用ABCA4样品对这种重要的光感受器细胞蛋白进行结构解析。
