Preparative refolding of small monomeric outer membrane proteins.
The outer membrane of gram-negative bacteria constitutes an important hurdle for the transport of hydrophobic molecules into the cell. Mass flux is often facilitated by various outer membrane proteins. These proteins are of biotechnological importance because they could help to improve the performance of whole-cell biocatalysts or be incorporated into artificial cell-like systems. The characterization and understanding of their transport properties greatly benefits from the possibility to express and purify these proteins. We investigated folding parameters for the refolding of four small monomeric outer membrane proteins from Escherichia coli (OmpW) and different pseudomonads (AlkL, OprG and TodX). To this aim we screened a number of inexpensive detergents and detergent concentrations, folding additives as well as protein concentrations. Interestingly, detergents with a C12chain were most effective in promoting the folding reaction, particularly the negatively charged N-Lauroylsarcosine for OmpW, OprG and TodX as well as the zwitterionic N,N-Dimethyl-n-dodecylamine N-oxide (LDAO) for AlkL. The addition of 1 M urea (AlkL, OmpW), 0.1 M glutamate (OprG) or 0.1 M glycine (TodX) could further improve the folding efficiency. In order to be able to reproducibly produce larger amounts of the proteins, we then established the folding in a miniaturized stirred-tank reactor system combined with a liquid handler. This approach led to a near-complete refolding of OprG (96%), a very good folding of AlkL (84%) and OmpW (71%), only TodX folding was more variable with a final folding efficiency of 52%, all obtained at a final protein concentration of 0.5 g/L.
Institute of Biochemical Engineering, Technical University of Munich, Boltzmannstraße 15, D-85748 Garching, Germany.
Tom Sebastian
TS
Schwarzer
Institute of Biochemical Engineering, Technical University of Munich, Boltzmannstraße 15, D-85748 Garching, Germany.
Maria
M
Hermann
Chair of Systems Biophysics and Bionanotechnology, Technical University of Munich, Am Coulombwall 4a, D-85748 Garching, Germany.
Swati
S
Krishnan
Chair of Systems Biophysics and Bionanotechnology, Technical University of Munich, Am Coulombwall 4a, D-85748 Garching, Germany.
Friedrich C
FC
Simmel
Institute of Biochemical Engineering, Technical University of Munich, Boltzmannstraße 15, D-85748 Garching, Germany. Electronic address: k.castiglione@lrz.tum.de.
Kathrin
K
Castiglione
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D058849:Protein Refolding; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D016958:Pseudomonas putida / Q000737:chemistry*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C121501:OprG protein, Pseudomonas aeruginosa; C092771:TodX protein, Pseudomonas putida; C064035:ompW protein, E coli
AlkL; Detergents; Inclusion bodies; Outer membrane proteins; Refolding