N. Yu. Mamaeva, N. I. Derkacheva, D. A. Gasanova, O. S. Sokolova, G. S. Glukhov
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引用次数: 0
Abstract
A detergent-free protocol for purification of the coronavirus prefusion S-protein using styrene-maleic acid copolymer (SMA) was developed. Expression of the S-protein was carried out in HEK293T cells. Two solubilization methods were used to purify and prepare the S-protein for microscopy: in NP-40 detergent and as part of SMA. The resulting preparations were examined in an electron microscope, and the particles of purified S-proteins were classified. Analysis of two-dimensional projections of the particles showed that the use of lipodiscs for solubilization leads to lower mobility of the purified protein on the substrate compared to the protein in the detergent, which may further contribute to obtaining higher resolutions when studying the structure of membrane proteins.
摘要 利用苯乙烯-马来酸共聚物（SMA）开发了一种纯化冠状病毒预融合 S 蛋白的无洗涤剂方案。S 蛋白在 HEK293T 细胞中表达。使用两种溶解方法纯化和制备用于显微镜观察的 S 蛋白：在 NP-40 去污剂中和作为 SMA 的一部分。在电子显微镜下对制备结果进行检查，并对纯化的 S 蛋白颗粒进行分类。对颗粒二维投影的分析表明，与洗涤剂中的蛋白质相比，使用脂溶盘进行增溶会降低纯化蛋白质在基质上的流动性，这可能有助于在研究膜蛋白结构时获得更高的分辨率。
期刊介绍：
Moscow University Biological Sciences Bulletin is forum for research in all important areas of modern biology. It publishes original work on qualitative, analytical and experimental aspects of research. The scope of articles to be considered includes plant biology, zoology, ecology, evolutionary biology, biophysics, genetics, genomics, proteomics, molecular biology, cell biology, biochemistry, endocrinology, immunology, physiology, pharmacology, neuroscience, gerontology, developmental biology, bioinformatics, bioengineering, virology, and microbiology.
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