Preparative refolding of small monomeric outer membrane proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Outer membrane proteins (OMPs) from gram-negative bacteria are crucial for nutrient uptake but pose challenges for hydrophobic molecule transport.
• These OMPs are of significant biotechnological interest for applications in whole-cell biocatalysis and artificial cell systems.
• Efficient expression and purification of OMPs are essential for understanding their transport functions.

Purpose of the Study:

• To investigate and optimize folding parameters for refolding small, monomeric OMPs from Escherichia coli and pseudomonads.
• To identify effective detergents, additives, and concentrations for OMP refolding.
• To establish a scalable method for reproducible OMP production.

Main Methods:

• Screening of various inexpensive detergents and detergent concentrations.
• Testing of different folding additives and protein concentrations.
• Implementation of a miniaturized stirred-tank reactor system combined with a liquid handler for scalable refolding.

Main Results:

• C12-chain detergents, specifically N-Lauroylsarcosine and N,N-Dimethyl-n-dodecylamine N-oxide (LDAO), proved most effective for refolding.
• Additives like urea, glutamate, and glycine significantly improved folding efficiency for specific OMPs.
• Scalable refolding yielded high efficiencies: OprG (96%), AlkL (84%), OmpW (71%), and TodX (52%) at 0.5 g/L protein concentration.

Conclusions:

• Optimized refolding protocols using specific C12 detergents and additives enable efficient production of OMPs.
• The developed reactor system facilitates reproducible, large-scale production of functional OMPs.
• These findings support the use of OMPs in biotechnological applications, enhancing whole-cell biocatalysts and artificial cell systems.