1. Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach.
- Author
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Katzen F, Fletcher JE, Yang JP, Kang D, Peterson TC, Cappuccio JA, Blanchette CD, Sulchek T, Chromy BA, Hoeprich PD, Coleman MA, and Kudlicki W
- Subjects
- Antiporters biosynthesis, Antiporters chemistry, Antiporters genetics, Apolipoprotein A-I biosynthesis, Apolipoprotein A-I chemistry, Apolipoprotein A-I genetics, Apolipoprotein E4 biosynthesis, Apolipoprotein E4 chemistry, Apolipoprotein E4 genetics, Bacteriorhodopsins biosynthesis, Bacteriorhodopsins chemistry, Bacteriorhodopsins genetics, Chromatography, Gel, Escherichia coli Proteins biosynthesis, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Humans, Membrane Proteins biosynthesis, Membrane Proteins genetics, Microscopy, Atomic Force, Solubility, Lipid Bilayers chemistry, Membrane Proteins chemistry
- Abstract
We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets.
- Published
- 2008
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