Department of Chemistry, Lehigh University, 6 E. Packer Ave. Bethlehem, Pennsylvania, 18015, USA.
Department of Chemistry, Lehigh University, 6 E. Packer Ave. Bethlehem, Pennsylvania, 18015, USA.
Anal Biochem. 2022 Feb 1;638:114497. doi: 10.1016/j.ab.2021.114497. Epub 2021 Nov 27.
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.
制备纳米盘的第一步是表达和纯化膜支架蛋白（MSP）至均一性。目前用于 MSP 分离和纯化的方法利用镍亲和层析。然而，MSP 上存在多组氨酸标签常常会干扰下游步骤，其中需要从空纳米盘中分离出与蛋白质重组的纳米盘。因此，在形成纳米盘之前，必须使用蛋白酶从 MSP 上去除多组氨酸标签，这是一个繁琐的过程。在此，我们描述了一种通过表达为包涵体，然后用溴化氰切割，凝胶过滤层析纯化，制备无标签 MSP 的稳健简化方法。此外，所制备的 MSP 不含色氨酸残基，这有利于基于色氨酸的重组蛋白的光谱研究。动态光散射和透射电子显微镜显示，所制备的无标签 MSP 能够产生纳米盘。
