CELL ENVELOPE PREPARATION / SOLUBILIZATION

1. CELL ENVELOPE PREPARATION / SOLUBILIZATION Kris-itd.unair.ac.id (for education purpose only)

2. Method : Grow bacteria in 5ml Proteoze Peptone No2 broth (for Pseudomonas) or Luria broth (10g tryptone, 5g yeast extract, 5g Nacl per liter; for E. coli) overnight at 37ºC with appropriate antibiotics. (Optional for larger prep.) Inoculate 100 ml of the corresponding broth with the overnight culture. Grow at 37oC until an OD600 0.6 to 0.8 is reached (or overnight depending on application). Centrifuge at 7,000 rpm for 10 minutes and resuspend the pellet in 10 mM Na2HPO4, 5 mM MgSO4, pH 7.4. For E. coli, resuspend in 10 mMTris-HCl, 5 mM MgSO4, pH 7.4 and add Triton X-100 to a concentration of 2%, then sonicate before proceeding to step 6. NOTE: Stocks of Na2HPO4 and MgSO4 solutions must be autoclaved separately. Add DNase to a concentration of 50 µg/ml and French press twice at 15,000 psi. (See French Press protocol in this manual). Centrifuge at 3,000 rpm for 10 minutes to remove cell debris. Decant the supernatant into a 70.1 Ti tube and centrifuge at 45,000 rpm for 1 hour.

3. Resuspend the pellet (cell envelope) in distilled water. For E. coli outer membranes, adjust envelope prep. in water to 10 mM Tris-HCl, 5 mM MgSO4, pH 7.4 and add Triton X-100 to a concentration of 2%. (Be sure protein concentration is less than 10 mg/ml.) Transfer to 70.1 Ti ti tube and fill tube with buffer from step 8, if necessary. Spin at 45,000 rpm for one hour in a 70.1 Ti. Resuspend the pellet (E. coli outer membrane) in distilled water. To enrich prep for certain porins, bring prep to 2% SDS, 10 mM Tris-HCl pH 7.4 (no EDTA). Incubate at 40ºC. for 10 min. Sonicate to get proper disruption. The protein concentration is less than 10 mg/ml. This should solubilize non-peptidoglycan associated proteins. Pellet as in step 9, using buffer from step 11. Repeat step 11 adding EDTA (10 mM EDTA) to the buffer (for OmpF use 0.2M NaCl also). This should solubilize peptidoglycan associated proteins. Pellet as in step 9 but reduce volume, using buffer from step 13. Using the bench top ultracentrifuge (Cellulase group) and the TLA100.2 Rotor (Tufaru's Lab) you can get the proper Xg force in less than one ml. Protein concentration should be at 10 mg/ml. SAVE supernatant. Porins should be in supernatant, and the peptidoglycan in pellet.