Applied and Industrial Microbiology Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, 600 036, India.
Sci Rep. 2020 Oct 1;10(1):16334. doi: 10.1038/s41598-020-73522-1.
Membrane protein purification is a laborious, expensive, and protracted process involving detergents for its extraction. Purifying functionally active form of membrane protein in sufficient quantity is a major bottleneck in establishing its structure and understanding the functional mechanism. Although overexpression of the membrane proteins has been achieved by recombinant DNA technology, a majority of the protein remains insoluble as inclusion bodies, which is extracted by detergents. Detergent removal is essential for retaining protein structure, function, and subsequent purification techniques. In this study, we have proposed a new approach for detergent removal from the solubilized extract of a recombinant membrane protein: human phospholipid scramblase 3 (hPLSCR3). N-lauryl sarcosine (NLS) has been established as an effective detergent to extract the functionally active recombinant 6X-his- hPLSCR3 from the inclusion bodies. NLS removal before affinity-based purification is essential as the detergent interferes with the matrix binding. Detergent removal by adsorption onto hydrophobic polystyrene beads has been methodically studied and established that the current approach was 10 times faster than the conventional dialysis method. The study established the potency of polystyrene-based beads as a convenient, efficient, and alternate tool to dialysis in detergent removal without significantly altering the structure and function of the membrane protein.
膜蛋白的纯化是一项费力、昂贵且耗时的过程，需要使用去污剂进行提取。在建立其结构和理解功能机制方面，大量获得具有功能活性的膜蛋白形式是一个主要的瓶颈。尽管通过重组 DNA 技术已经实现了膜蛋白的过表达，但大多数蛋白质仍然以包涵体的形式不溶，需要用去污剂提取。去污剂的去除对于保留蛋白质的结构、功能以及随后的纯化技术是必不可少的。在这项研究中，我们提出了一种从重组膜蛋白人磷脂翻转酶 3（hPLSCR3）的可溶提取物中去除去污剂的新方法。N-月桂酰肌氨酸（NLS）已被确立为一种有效的去污剂，可从包涵体中提取具有功能活性的重组 6X-his-hPLSCR3。在基于亲和性的纯化之前去除 NLS 是必要的，因为去污剂会干扰基质结合。已经系统地研究了通过吸附到疏水性聚苯乙烯珠上去除去污剂，并确定当前方法比传统的透析方法快 10 倍。该研究确立了聚苯乙烯珠作为一种方便、高效的替代透析方法的潜力，可用于去除去污剂，而不会显著改变膜蛋白的结构和功能。
