Materials and Reagents

125 mL flask (PYREX, catalog number: 4980)
2,800 mL flask (PYREX, catalog number: 4420)
Pipette tips
Serological pipets
1 L centrifuge bottle (Thermo Scientific, Nalgene^TM, catalog number: 3141-1006)
70 mL ultracentrifuge bottle (Beckman Coulter, catalog number: 355622)
37 mL ultracentrifuge tube (Beckman Coulter, catalog number: 344058)
Tissue grinder (DWK Life Sciences, catalog number: 8853030040)
Luria broth (Research Products International, catalog number: L24400-5000.0)
Terrific broth (Research Products International, catalog number: T15100-5000.0)
Agar (BD, Bacto^TM, catalog number: 214010)
Tris (American Bio, catalog number: AB02000-05000)
Sodium chloride (NaCl) (Dot Scientific, catalog number: DSS23020-5000)
Glycerol (J.T. Baker, catalog number: 2136-03)
Kanamycin sulfate (American Bio, catalog number: AB01100-00010)
Spectinomycin (Research Products International, catalog number: S23000-25.0)
Chloramphenicol (Sigma, catalog number: C0378-25G)
Sucrose (Sigma-Aldrich, catalog number: S0389-1 KG)
IPTG (isopropyl β-d-1-thiogalactopyranoside) (American Bio, catalog number: AB00841-00010)
PMSF (phenylmethylsulfonyl fluoride) (Dot Scientific, catalog number: DSP20270-25)
2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250-100ML)
Glycerol stock of BL21 (DE3) star plysS strain expressing the YejM/LapB complex (Shu and Mi, 2022)
Hydrochloric acid (J.T. Baker, catalog number: 9535-00)
LB medium (see Recipes)
LB agar plate (see Recipes)
TB medium (see Recipes)
1 M Tris-HCl, pH 7.8 (see Recipes)
Lysis buffer (see Recipes)
Dilution buffer (see Recipes)
73% sucrose (see Recipes)
53% sucrose (see Recipes)
20% sucrose (see Recipes)

Equipment

Pipettes (P200, P1000)
Pipette controller (BrandTech Scientific, model: accu-jet pro)
-80 °C ultra-low temperature freezer (PHCbi, model: MDF-U76VA-PA)
Microbiological incubator (VWR, model: F Air 6.3 CF)
Incubator shaker (New Brunswick, model: Innova^TM 44)
Centrifuge (Thermo Scientific Sorvall, model: BP8)
Ultracentrifuge (Beckman, model: Optima^TM LE-80K)
High-pressure microfluidizer (Avestin, model: Emulsi Flex-C3)
Fixed angle rotor (Beckman Coulter, model: Type 45Ti)
Swinging-bucket rotor (Beckman Coulter, model: SW 28)
Ultrapure water system (Millipore, model: Reference A+)
pH meter (Apera, model: pH700)

Procedure

Prepare total cell membrane
1. Streak the glycerol stock of BL21 (DE3) star plysS strain expressing the YejM/LapB complex on an LB agar plate containing 50 µg/mL of kanamycin sulfate, 50 µg/mL of spectinomycin, and 25 µg/mL chloramphenicol (Kan⁺Spe⁺Cam⁺). Incubate the plate at 37 °C on a microbiological incubator overnight.
2. Inoculate a single colony from the overnight culturing plate into 20 mL of LB medium (Kan⁺Spe⁺Cam⁺) in a 125 mL flask. Grow the cells at 37 °C with shaking at 220 rpm overnight.
3. Transfer 10 mL of overnight culture to 1 L of TB medium (Kan⁺Spe⁺Cam⁺) in 2,800 mL flasks (2 L in total). Grow the cells at 37 °C with shaking at 220 rpm to OD₆₀₀ of 0.6.
4. Add 200 µL of 1 M IPTG to 1 L of cell culture (final concentration 0.2 mM) and incubate the cells at 30 °C with shaking at 220 rpm for another 4 h.
5. Centrifuge the culture at 5,000 × g for 10 min at 4 °C to harvest the cells.
6. Resuspend the cell pellet in 40 mL of lysis buffer (see Recipes) supplemented with 1 mM PMSF and 5 mM 2-Mercaptoethanol. At this step, resuspended cells can be flash-frozen in liquid nitrogen and stored in a -80 °C ultra-low temperature freezer, or move on directly to step A7 to break the cells.
7. Transfer the cells to a 40 mL tissue grinder. Homogenize the cells by pushing and pulling the grind pestle 10–20 times.
8. Break the cells in a high-pressure microfluidizer at 15,000 psi with two passes.
9. Transfer the broken cells to 50 mL centrifuge tubes and remove cell debris by centrifugation at 5,000 × g for 10 min at 4 °C.
10. Transfer the supernatant to a 70 mL ultracentrifuge bottle and add lysis buffer to the final volume of ~50 mL. Carefully balance the ultracentrifuge bottles.
11. Ultracentrifuge in a Beckman Coulter type 45Ti rotor at 40,000 rpm (185,677 × g) for 2 h at 4 °C. Keep the membrane pellet after centrifugation.

Separate inner and outer membranes
1. Resuspend ~3 g of membrane in 55 mL of 20% sucrose with a 40 mL tissue grinder.
2. Prepare six 37 mL ultracentrifuge tubes. In each tube, from the bottom to the top, layer 14 mL of 73% sucrose, 14 mL of 53% sucrose, and 9 mL of membrane resuspended in 20% sucrose.
3. Ultracentrifuge without braking at 23,000 rpm (65,000 × g) in a SW 28 rotor for 18 h at 4 °C.
4. After separation, there should be two bands: the upper band is the IM and the lower one is the OM (Figure 1).
  
  Figure 1. Separation of IM and OM
5. Cut the end of a P1000 pipette tip, approximately 5 mm from the point. Collect the upper IM layer using the pipette.
6. Use a new pipette tip and repeat step B5 to collect the OM layer.
7. To remove sucrose from the membrane, transfer the IM and OM fractions into 70 mL ultracentrifuge bottles. Add dilution buffer into the bottles to a final sucrose concentration of <10%. Carefully balance the ultracentrifuge bottles.
8. Ultracentrifuge in a Beckman Coulter type 45Ti rotor at 40,000 rpm (185,677 × g) for 2 h at 4 °C.
9. Discard the supernatant and weigh the wet weight of the membrane. For 1 g of the membrane, add 10 mL of lysis buffer to resuspend using a 40 mL tissue grinder.
10. Flash-freeze the membrane with liquid nitrogen and store in a -80 °C ultra-low temperature freezer.

Notes

This protocol uses E. coli strain BL21 (DE3) star plysS expressing the YejM/LapB complex as an example to separate IM and OM. This protocol also applies to other E. coli strains.
All the buffers used in this protocol are kept at 4 °C.
When separating the IM and OM, for each 37 mL ultracentrifuge tube, do not add more than 0.5 g of the total membrane. Otherwise, the IM and OM may not be separated well.
To remove the sucrose in the membrane by ultracentrifuge, the membrane must be first diluted to a final sucrose concentration of less than 10%.

Recipes

LB medium
25 g Luria broth, dissolve in 1 L of deionized water. Autoclave at 121 °C for 30 min.

LB agar plate
LB medium with 1.5% w/v agar.

TB medium
47.6 g Terrific broth and 4 mL of glycerol. Dissolve in 1 L of deionized water.
Autoclave at 121 °C for 30 min.

1 M Tris-HCl, pH 7.8
Dissolve 121.14 g of Tris in 800 mL of Milli-Q water. Adjust the pH to 7.8 using hydrochloric acid. Then add Milli-Q water to a final volume of 1 L.

Lysis buffer
50 mM Tris-HCl, pH 7.8
300 mM NaCl
10% glycerol (v/v)

Dilution buffer
50 mM Tris-HCl, pH 7.8
300 mM NaCl

73% sucrose
73% w/v sucrose
20 mM Tris-HCl, pH 7.8

53% sucrose
53% w/v sucrose
20 mM Tris-HCl, pH 7.8

20% sucrose
20% w/v sucrose
20 mM Tris-HCl, pH 7.8

Acknowledgments

Research reported in this publication was supported by NIAID and NIGMS of the National Institutes of Health under award numbers R21AI156595 and R01GM137068. This protocol was previously used in our reported results (Shu and Mi, 2022).

Competing interests

S. S and W. M declare no conflicts of interest.

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