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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.
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- 2 Finished
1/10/14 → 28/02/18
1/01/12 → 1/01/16