High level cell-free expression and specific labeling of integral membrane proteins.
We demonstrate the high level expression of integral membrane proteins (IMPs) in a cell-free coupled transcription/translation system using a modified Escherichia coli S30 extract preparation and an optimized protocol. The expression of the E. coli small multidrug transporters EmrE and SugE containing four transmembrane segments (TMS), the multidrug transporter TehA with 10 putative TMS, and the cysteine transporter YfiK with six putative TMS, were analysed. All IMPs were produced at high levels yielding up to 2.7 mg of protein per mL of reaction volume. Whilst the vast majority of the synthesized IMPs were precipitated in the reaction mixture, the expression of a fluorescent EmrE-sgGFP fusion construct showed evidence that a small part of the synthesized protein 'remained soluble and this amount could be significantly increased by the addition of E. coli lipids into the cell-free reaction. Alternatively, the majority of the precipitated IMPs could be solubilized in detergent micelles, and modifications to the solubilization procedures yielded proteins that were almost pure. The folding induced by formation of the proposed alpha-helical secondary structures of the IMPs after solubilization in various micelles was monitored by CD spectroscopy. Furthermore, the reconstitution of EmrE, SugE and TehA into proteoliposomes was demonstrated by freeze-fracture electron microscopy, and the function of EmrE was additionally analysed by the specific transport of ethidium. The cell-free expression technique allowed efficient amino acid specific labeling of the IMPs with 15N isotopes, and the recording of solution NMR spectra of the solubilized EmrE, SugE and YfiK proteins further indicated a correctly folded conformation of the proteins.
Klammt
Christian
C
Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main, Institute for Biophysical Chemistry, Frankfurt/Main, Germany.
Löhr
Frank
F
Schäfer
Birgit
B
Haase
Winfried
W
Dötsch
Volker
V
Rüterjans
Heinz
H
Glaubitz
Clemens
C
Bernhard
Frank
F
D001483:Base Sequence; D002474:Cell-Free System; D002942:Circular Dichroism; D017931:DNA Primers; D003902:Detergents; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D005614:Freeze Fracturing; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D008854:Microscopy, Electron; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011510:Proteolipids
D017931:DNA Primers; D003902:Detergents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011510:Proteolipids; C020485:proteoliposomes