MATERIALS:

• Proteose Peptone #2 (PP2)
• 1M Tris-HCl, pH 8.0
• 0.5M EDTA, pH 8.0
• Triton X-100
• Deoxyribonuclease I
• Sucrose (20%, 52%, 70%)

METHOD:

1. Grow H103 in 100ml PP2 in a 2 litre flask overnight at 37ºC. Innoculate into 2 litres of PP2 in 6 a litre flask the next day. Grow until an OD₆₀₀ = 0.6 - 0.8.
2. Harvest cells at 7K for 15 minutes. Resuspend the pellet in 20% sucrose, 10 mM Tris-HCl, pH 8.0.
3. Add 10ml of DNase I (50 µg/ml). French press at 15,000 psi three times. Centrifuge the cell lysate at 3,000 rpm for 10 minutes.
4. Set up a 2-step sucrose gradient (52% and 72%). Apply the sample on top of the gradient. Centrifuge at 21,000 rpm overnight in an SW 27 rotor.
5. Collect the outer membrane band by poking a hole at the bottom of the tube and dripping the fraction into a 60Ti tube. (The band closest to the bottom of the tube)
6. Dilute the fraction with distilled water to fill the 60Ti tube. Centrifuge at 45,000 rpm for 1 hour.
7. Resuspend the pellet in distilled water. Perform a protein assay.
8. Perform the solubilization as follows:


2 X in 2% TX 100, 20mM Tris-HCL, pH 8.0

3 X in 2% TX 100, 20mM Tris-HCl, pH 8.0, 10mM EDTA
 

9. Sonicate at a probe intensity of 35 for two 30 second bursts and centrifuge at 45,000 rpm for 1 hour for each of the solubilizations. Save all the fractions.
10. Resuspend the pellet in distilled water. Perform a KDO assay and a protein assay. Perform gel electrophoresis on all fractions.
11. Add 1mg/ml lysozyme to the OprF containing fractions and let stand for 10 minutes. Solubilize with 2% TX 100, 20mM Tris-HCl, pH 8.0.
12. Save the supernatant and pellet. Pass the supernatant over a DEAE-Sephacel column equilibrated with 0.2% TX 100, 20mM Tris-HCl, pH 8.0, 10 mM EDTA, pH 8.0 (column buffer).
13. Perform the column chromatography as follows:


Prepare a slurry of DEAE-Sephacel by washing several times with distilled water, then column buffer. Deaerate and pour into a 30 ml column. After column is packed, equilibrate it with at least five bed volumes of column buffer.

Apply the sample by the dry bed method.

Elute the porin F in three stages, two bed volumes of column buffer, two bed volumes of column buffer with 0.1M NaCl and two bed volumes of column buffer with 0.3M NaCl.

Collect 1.0 ml fractions.
 

14. Perform a protein assay on each fraction and gel electrophoresis on major peaks to determine where OprF is.