Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach.
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.
Katzen
Federico
F
Invitrogen Corporation, Carlsbad, CA 92008, USA.
Fletcher
Julia E
JE
Yang
Jian-Ping
JP
Kang
Douglas
D
Peterson
Todd C
TC
Cappuccio
Jenny A
JA
Blanchette
Craig D
CD
Sulchek
Todd
T
Chromy
Brett A
BA
Hoeprich
Paul D
PD
Coleman
Matthew A
MA
Kudlicki
Wieslaw
W
D017920:Antiporters / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016632:Apolipoprotein A-I / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D053327:Apolipoprotein E4 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001436:Bacteriorhodopsins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D002850:Chromatography, Gel; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D018625:Microscopy, Atomic Force; D012995:Solubility
C505693:APOA1 protein, human; D017920:Antiporters; D016632:Apolipoprotein A-I; D053327:Apolipoprotein E4; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; C075436:EmrE protein, E coli; D001436:Bacteriorhodopsins