Li Dianfan, Caffrey Martin
School of Biochemistry and Immunology & School of Medicine, Trinity College Dublin, Dublin, Ireland.
Sci Rep. 2014 Jul 24;4:5806. doi: 10.1038/srep05806.
Membrane proteins play vital roles in the life of the cell and are important therapeutic targets. Producing them in large quantities, pure and fully functional is a major challenge. Many promising projects end when intractable aggregates or precipitates form. Here we show how such unfolded aggregates can be solubilized and the solution mixed with lipid to spontaneously self-assemble a bicontinuous cubic mesophase into the bilayer of which the protein, in a confined, chaperonin-like environment, reconstitutes with 100% efficiency. The test protein, diacylglycerol kinase, reconstituted in the bilayer of the mesophase, was then crystallized in situ by the in meso or lipid cubic phase method providing an X-ray structure to a resolution of 2.55 Å. This highly efficient, inexpensive, simple and rapid approach should find application wherever properly folded, membrane reconstituted and functional proteins are required where the starting material is a denatured aggregate.
膜蛋白在细胞生命活动中发挥着至关重要的作用，是重要的治疗靶点。大量生产纯净且功能完备的膜蛋白是一项重大挑战。许多前景良好的项目在形成难以处理的聚集体或沉淀时就宣告结束。在此，我们展示了如何使此类未折叠的聚集体溶解，并将溶液与脂质混合，使其自发自组装成双连续立方中间相，蛋白质在受限的、类似伴侣蛋白的环境中以100%的效率在中间相的双层中重构。测试蛋白二酰基甘油激酶在中间相的双层中重构后，通过“in meso”或脂质立方相方法原位结晶，得到分辨率为2.55 Å的X射线结构。这种高效、廉价、简单且快速的方法应能在任何需要以变性聚集体为起始材料来获得正确折叠、膜重构且功能正常的蛋白质的地方得到应用。
