Facile production of tagless membrane scaffold protein for nanodiscs.
Facile production of tagless membrane scaffold protein for nanodiscs.
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DOI：
10.1016/j.ab.2021.114497
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发表时间：
2022-02-01
影响因子：
2.9
通讯作者：
Glover KJ
中科院分区：
文献类型：
--
作者：
Julien JA;Mutchek SG;Fernandez MG;Glover KJ
制备纳米盘的第一步是表达和纯化膜支架蛋白（MSP）至均一。目前用于分离和纯化MSP的方法利用镍亲和层析。然而，MSP上多组氨酸标签的存在经常干扰下游步骤，其中需要将用蛋白质重构的纳米盘与空纳米盘分离。因此，在形成纳米盘之前，必须进行使用蛋白酶从MSP去除聚组氨酸标签的挑剔过程。在此，我们描述了一个强大的流线型的方法来生产无标签MSP表达的包涵体，然后用溴化氰裂解，并通过凝胶过滤色谱纯化。此外，所制备的MSP不含色氨酸残基，这有利于重构蛋白质的基于色氨酸的光谱研究。动态光散射和透射电子显微镜显示，所产生的无标签MSP能够产生纳米盘。
The initial step in the preparation of nanodiscs is to express and purify the membrane scaffold protein (MSP) to homogeneity. Current methods used for the isolation and purification of MSP utilize nickel affinity chromatography. However, the presence of a polyhistidine tag on the MSP often interferes with downstream steps where nanodiscs reconstituted with protein need to be isolated from empty ones. Therefore, one must engage in the finicky process of removing the polyhistidine tag from the MSP using a protease before the formation of nanodiscs. Herein, we describe a robust streamlined approach to produce tagless MSP by expression as inclusion bodies followed by cleavage with cyanogen bromide, and purification by gel filtration chromatography. In addition, the MSP prepared is devoid of tryptophan residues which facilitates tryptophan-based spectroscopic studies of reconstituted proteins. Dynamic light scattering and transmission electron microscopy showed that the tagless MSP produced was competent to produce nanodiscs.
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