Here is a protocol published by a colleague of mine. This protocol results in preparation of outer membranes with the proteins remaining in the membrane. The outer membranes can be dissolved using n-octylglucoside, a nonionic detergent that may allow the proteins to remain in their native structure, to release the proteins.

Overnight cultures were spun down at

5000g for 10 min at 4 °C, resuspended in 10 mM HEPES

buffered at pH 7.5, and lysed using a French Press cell (Glen

Mills, Clifton, NJ) at 16 000−18 000 psi. The lysate was

clarified by low-speed centrifugation (10 000g, 20 min, 8 °C)

to remove unlysed cells and debris and centrifuged to isolate

total bacterial membranes (58 000g, 1.5 h, 8 °C). The inner

membrane lipids and proteins were solubilized (35 °C, 35 min,

240 rpm) with 1% N-lauroylsarcosine (Millipore Sigma, St.

Louis, MO), and outer membranes were isolated by ultracentrifugation

(58 000g, 1.5 h, 8 °C). Outer membrane pellets

were resuspended gently with a 21-gauge needle and 1 mL

hypodermic syringe, and total protein was quantified using the

Pierce BCA protein assay kit (ThermoFisher, Waltham, MA).

Samples were typically stored at −80 °C until further use.

Iyer et al. Acinetobacter baumannii OmpA Is a Selective Antibiotic Permeant

Porin. ACS Infect. Dis. 2018, 4, 373−381

Dear JAsmine

a possible alternative is the isolation of Outer membrane vesicles from E.coli surnatant (it can be done using ultracentrifugation) which may contain the most of outer membrane proteins which are also present in the bacterial surface.

Microbial biosynthesis of designer outer membrane vesicles (nih.gov)

The productivity of standard wild type E.coli strain ( Membrane Vesicles Released by a hypervesiculating Escherichia coli Nissle 1917 tolR Mutant Are Highly Heterogeneous and Show Reduced Capacity for Epithelial Cell Interaction and Entry (storage.googleapis.com) could be low but if you do not need an high amount of sample you can try this approach.

With Delta TolR strain, several years ago by replacing growth in the LB medium at 37°C with the YGR high cell density medium at 30°C we were able to acheive about to fold improvment in vesicle productivity

You can find more information about YGR medium at the following links

https://www.blogger.com/video.g?token=AD6v5dzLWqhoYwVoIC07Aj1G4RRYNxwM3Ndyk09KE3rmvGIdgfhR6WOBAH6TE0sDThwNquI9hZ9u7vKUDdMsusqQ3IcouXA53Y37DqhPmlYX4e9_Jnz5YS-87K68gQcCTjBMvNE4MlI

Sincerely i never applied it in standard E.coli wild type strains, but it may work well. Of course if you are pass through OMV production you have to set up some vesicles quality control step to be sure that you are producing vesicles and not simple bacterial debris.

good luck

MAnuele

I think the best preparation of E. coli OM is by Darveau et al, circa 1982 or 1983. I would stay away from all of the many detergent extraction techniques as they do not work all that well. For a really clean prep see Darveau.