A modular approach for balanced overexpression of recombinant multiprotein complexes in E. coli is described, with the prokaryotic protein secretase/insertase complex, the SecYEG-SecDFYajC-YidC holotranslocon (HTL), used as an example. This procedure has been implemented here in the ACEMBL system. The protocol details the design principles of the monocistronic or polycistronic DNA constructs, the expression and purification of functional HTL and its association with translating ribosome nascent chain (RNC) complexes into a RNC-HTL supercomplex.
Multiprotein complexes Membrane proteins Protein insertion and secretion Holotranslocon HTL ACEMBL system Cre recombinase Donor–acceptor fusion Subunit stoichiometry Ribosome nascent chain complex RNC
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We thank all members of the Berger, Schaffitzel, and Collinson laboratories for helpful discussions. The authors thank Sir John Walker for the E. coli C43 expression strain. J.K. was supported by a doctoral training grant from the BBSRC. C.S. is supported by a European Research Council ERC Starting Grant Award. I.C. acknowledges support by the BBSRC (Project Grants BB/M003604/1 and BB/I008675/1) and is recipient of a Wellcome Trust Senior Investigator Award. I.B. acknowledges support from the European Commission Framework Programme 7 ComplexINC project (contract no. 279039).
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