Komar Joanna, Botte Mathieu, Collinson Ian, Schaffitzel Christiane, Berger Imre
School of Biochemistry, University of Bristol, Bristol, United Kingdom.
European Molecular Biology Laboratory, Grenoble, France; Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Unité mixte de Recherche, Grenoble, France.
Methods Enzymol. 2015;556:23-49. doi: 10.1016/bs.mie.2014.12.027. Epub 2015 Mar 20.
Membrane proteins constitute about one third of the proteome. The ubiquitous Sec machinery facilitates protein movement across or integration of proteins into the cytoplasmic membrane. In Escherichia coli post- and co-translational targeting pathways converge at the protein-conducting channel, consisting of a central pore, SecYEG, which can recruit accessory domains SecDF-YajC and YidC, to form the holotranslocon (HTL) supercomplex. Detailed analysis of HTL function and architecture remained elusive until recently, largely due to the lack of a purified, recombinant complex. ACEMBL is an advanced DNA recombineering-based expression vector system we developed for producing challenging multiprotein complexes. ACEMBL affords the means to combine multiple expression elements including promoter DNAs, tags, genes of interest, and terminators in a combinatorial manner until optimal multigene expression plasmids are constructed that yield correctly assembled, homogenous, and active multiprotein complex specimens. We utilized ACEMBL for recombinant HTL overproduction. We developed protocols for detergent solubilizing and purifying the HTL. Highly purified complex was then used to reveal HTL function and the interactions between its constituents. HTL activity in protein secretion and membrane protein insertion was analyzed in both the presence and absence of the proton-motive force. Setting up ACEMBL for the assembly of multigene expression constructs that achieve high yields of functional multisubunit membrane protein complex is straightforward. Here, we used ACEMBL for obtaining active HTL supercomplex in high quality and quantity. The concept can likewise be applied to obtain many other assemblies of similar complexity, by overexpression in prokaryotic, and also eukaryotic hosts.
膜蛋白约占蛋白质组的三分之一。普遍存在的Sec机制促进蛋白质跨细胞质膜的移动或蛋白质整合到细胞质膜中。在大肠杆菌中，翻译后和共翻译靶向途径在蛋白质传导通道处汇聚，该通道由中央孔SecYEG组成，它可以募集辅助结构域SecDF-YajC和YidC，形成全转运体（HTL）超复合物。直到最近，对HTL功能和结构的详细分析仍不清楚，这主要是由于缺乏纯化的重组复合物。ACEMBL是我们开发的一种先进的基于DNA重组工程的表达载体系统，用于生产具有挑战性的多蛋白复合物。ACEMBL提供了一种方法，以组合方式组合多个表达元件，包括启动子DNA、标签、感兴趣的基因和终止子，直到构建出能产生正确组装、同质且有活性的多蛋白复合物样本的最佳多基因表达质粒。我们利用ACEMBL过量生产重组HTL。我们开发了用于去污剂溶解和纯化HTL的方案。然后使用高度纯化的复合物来揭示HTL功能及其组成成分之间的相互作用。在有和没有质子动力势的情况下，分析了HTL在蛋白质分泌和膜蛋白插入中的活性。建立ACEMBL以组装能实现高产功能性多亚基膜蛋白复合物的多基因表达构建体很简单。在这里，我们使用ACEMBL高质量、高产量地获得了活性HTL超复合物。同样，通过在原核生物和真核生物宿主中过表达，这个概念也可以应用于获得许多其他类似复杂程度的组装体。
