Abstract
Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21 Gold (DE3) designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB), both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.
Highlights
The envelope of Gram-negative bacteria such as, Escherichia coli consists of two membranes, the inner and the outer membrane
To delete the ompF locus, we amplified the FLP recognition target (FRT)-kanamycin cassette flanked by sequence directly outside the sequence coding for OmpF, using the Keio collection ompF strain and the same primer sequences described in that study (Baba et al, 2006)
We have produced a series of E. coli knock-out strains for use in over-expressing OMP Production membrane proteins (OMPs) with deletions of at least one of four abundant OMP protein genes
Summary
The envelope of Gram-negative bacteria such as, Escherichia coli consists of two membranes, the inner and the outer membrane. Β-barrels consist of an antiparallel β-sheet that closes in on itself; the proteins adopt a cylindrical shape, with hydrophobic residues facing the membrane environment and mostly hydrophilic residues lining the inside of the β-barrel, which in the case of porins acts as an aqueous channel permitting the diffusion of water and other nutrients through the outer membrane (Delcour, 2009). Insertion of the kan cassette was achieved by λ red recombination, essentially as described (Datsenko and Wanner, 2000): the plasmid pKD46 was transformed into recipient strains by electroporation, and transformants were selected for by plating on ampicillin and growing at 30◦C. Insertion of the kan cassette into the ompF locus was verified by colony PCR
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