Stevens Charles M, Paetzel Mark
Department of Molecular Biology and Biochemistry, Simon Fraser University, South Science Building, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6.
Protein Expr Purif. 2012 Jul;84(1):167-72. doi: 10.1016/j.pep.2012.05.002. Epub 2012 May 15.
We present a method for the purification of the 45 residue long leader peptide of Escherichia coli dimethyl sulfoxide reductase subunit A (DmsA(L)), a substrate of the twin arginine translocase, by co-expressing the leader peptide with its specific chaperone protein, DmsD. The peptide can be isolated from the soluble DmsA(L)/DmsD complex or conveniently from the lysate pellet fraction. The recombinant leader peptide is functionally intact as the peptide/chaperone complex can be reconstituted from purified DmsA(L) and DmsD. A construct with DmsA(L) fused to the N-terminus of DmsD (DmsA(L)-DmsD fusion) was created to further explore the properties of the leader peptide-chaperone interactions. Analytical size-exclusion chromatography in-line with multi-angle light scattering reveals that the DmsA(L)-DmsD fusion construct forms a dimer wherein each protomer binds the neighboring leader peptide. A model of this homodimeric interaction is presented.
我们提出了一种纯化大肠杆菌二甲基亚砜还原酶亚基A（DmsA(L)）45个残基长的前导肽的方法，该前导肽是双精氨酸转运酶的底物，通过将前导肽与其特异性伴侣蛋白DmsD共表达来实现。该肽可以从可溶性DmsA(L)/DmsD复合物中分离出来，或者方便地从裂解物沉淀部分中分离出来。重组前导肽功能完整，因为肽/伴侣复合物可以由纯化的DmsA(L)和DmsD重构而成。构建了一个将DmsA(L)融合到DmsD N端的构建体（DmsA(L)-DmsD融合体），以进一步探索前导肽-伴侣相互作用的特性。与多角度光散射联用的分析尺寸排阻色谱显示，DmsA(L)-DmsD融合构建体形成二聚体，其中每个原体结合相邻的前导肽。本文给出了这种同二聚体相互作用的模型。
