A New Strain Collection for Improved Expression of Outer Membrane Proteins

Area of Science:

• Microbiology and Molecular Biology
• Protein Expression and Purification
• Structural Biology

Background:

• Outer membrane proteins (OMPs) in Gram-negative bacteria, primarily β-barrel proteins, are crucial for cellular functions and are significant targets for drug and vaccine development.
• Over-expression and purification of these membrane proteins for research, especially structural studies, present considerable challenges.
• Current methods often struggle to achieve high yields and purity of recombinant OMPs.

Purpose of the Study:

• To engineer novel *E. coli* BL21 Gold (DE3) deletion mutant strains specifically designed for enhanced over-expression of recombinant outer membrane proteins.
• To assess the utility of these mutant strains for producing various types of outer membrane proteins, including β-barrel proteins and autotransporters.
• To provide improved tools for biochemical and structural investigations of OMPs and facilitate biophysical measurements.

Main Methods:

• Generation of *E. coli* BL21 Gold (DE3) strains with deletions in genes encoding abundant outer membrane β-barrel proteins (OmpA, OmpC, OmpF, LamB).
• Construction of single, double, triple, and quadruple deletion mutants, ensuring complete gene deletion to prevent reversion.
• Expression testing of four different recombinant proteins (a small OMP, its variants, and two autotransporters) in the quadruple mutant strain compared to the parent strain.

Main Results:

• The quadruple deletion mutant strain demonstrated significantly improved expression levels and higher quality of the tested recombinant outer membrane proteins compared to the wild-type parent strain.
• Despite observed differences in growth and salt-induced aggregation, protein expression was not negatively impacted in the quadruple mutant under recommended handling conditions.
• The engineered strains are suitable for efficient production and purification of OMPs and are valuable for labeling experiments in native membrane environments.

Conclusions:

• The developed *E. coli* deletion mutant strains provide a robust platform for overcoming challenges in recombinant outer membrane protein over-expression and purification.
• These strains facilitate high-yield production of diverse OMPs, supporting advanced biochemical, structural, and biophysical studies.
• The engineered system offers a valuable resource for researchers targeting Gram-negative bacterial outer membrane proteins for therapeutic and diagnostic applications.