Cell-free production of integral membrane proteins on a preparative scale.
The chapter will focus on the high level cell-free production of integral membrane proteins having multiple transmembrane segments by using an individual coupled transcription/translation system based on an Escherichia coli S30-extract. We describe in detail the setup and optimization of the cell-free expression technique to obtain the maximum yield of recombinant proteins. The protocol can be used for the expression of soluble membrane proteins as well as for their production as a precipitate. In addition, we will provide protocols for the efficient solubilization and reconstitution of membrane proteins directly from the cell-free produced precipitates.
Klammt
Christian
C
Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main, Institute for Biophysical Chemistry, Frankfurt/Main, Germany.
Schwarz
Daniel
D
Dötsch
Volker
V
Bernhard
Frank
F
D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D003902:Detergents; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D005614:Freeze Fracturing; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D008954:Models, Biological; D008967:Molecular Biology / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure
D017920:Antiporters; D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins