Cell-free expression & assembly of a macromolecular membrane protein complex: a complete ATP synthase
Abstract
Cell-free expression technologies have emerged as promising methods for the production of individual membrane proteins. However, many membrane proteins need to be integrated in complex assemblies by interaction with soluble and membrane-bound subunits in order to adopt stable and functionally folded structures. The production of complete molecular machines by cell-free expression as advancement of the production of only individual subunits would open a variety of new possibilities to study their assembly mechanisms. We could demonstrate the successful cell-free formation of large molecular complexes consisting of both, membrane-bound and soluble subunits by expression of the 542-kDa ATP synthase from Caldalkalibacillus thermarum strain TA2.A1. The final complex is composed of 9 soluble and 16 membrane-embedded protomers. Full assembly into the functional complex was accomplished in different cell-free expression modes either by solubilizing initial precipitates, by cotranslational insertion into selected detergents or by cotranslational insertion into supplied liposomes. Assembly, membrane incorporation as well as specific activity of the complex was confirmed by biochemical analyses, freeze-fracture electron microscopy, single-particle analyses and immuno-gold labeling. This work establishes the production of highly complex molecular machines in defined environments either as proteomicelles or as proteoliposomes as a new application of cell-free expression systems.
