OmpF
Introduction
E. coli strain: MH225 (pPR272) or BL21- DE3, K-12 (needs to look for B5 or K-12)
“Expression of outer membrane proteins is also affected by medium pH; growth at low pH increases OmpC expression and decreases the level of OmpF. In contrast, the expression of OmpF was essentially unchanged at both high and low pHs” - Kobayashi (2000)
Expression
Cells are grown in LB broth at 37°C to an optical density, OD600≅1.5–2.0.
Harvest the cells (10,000xg for 20 mins) and wash the pellet with 30 mM Tris-Cl (pH 7.5) buffer.
Resuspend cell pellet (~1-1.5 g net weight) in 40 mL of cold buffer A (30 mM Tris-Cl (pH 7.5), 1 mM MgCl2, 1 mM PMSF).
French press 16,000 psi, centrifuge 2,000xg for 10 minutes, and collect the supernatant.
Add octyl-POE 0.5% (v/v) (29 mM final concentration) to the supernatant, and incubate 20 mins @ 20°C.
Centrifuge the extract @ 100,000xg for 1 hr. Discard the supernatant, and gently rinse the pellet with distilled water.
Resuspend pellet in 1mL of 3% (v/v) (172 mM) octyl-POE, 500 mM NaCl, 30 mM Tris-Cl (pH 7.5), 10 mM EDTA. Sonicate in the water bath for 2 min and incubate for 30 min @ 37°C.
Centrifuge for 15 mins @ 100,000xg, and keep the supernatant.
Purification
Load the sample (~20 mL) to the Superloop and then inject it onto Mono-Q HR 5/5 column, pre-equilibrated with (13 mM octyl-POE, 1 mM EDTA, 20 mM Tris-Cl, pH 7.5).
Elute the column with NaCl salt gradient from 500mM at 0.5 mL/min. (Buffer B: 13mM octyl-POE, 1 mM EDTA, 20 mM Tris-Cl, pH 7.5, 500 mM NaCl).
Load the peak fraction from the MonoQ onto a Superdex 200 column, equilibrated with 10mM octyl-POE, 75 mM NaCl, 20 mM Tris-Cl (pH 7.5), 0.05% NaN3, and 1 mM EDTA.
Collect peak fraction, and run the SDS-PAGE gels to characterize the eluted sample.
Note: The elution of OmpF is earlier than OmpC due to a net negative charge.
2D crystallization
Prepare dialysis membranes
Cut dialysis membrane tubing (Spectra/Por 12-14 kDa, Cat# 132678) into approximate length (~70 cm) and incubate them in Mili-Q water for 20 minutes.
Place in a 2-L beaker containing 50% EtOH, and bring to boil.
Boil for 60 mins. Notes that a smaller beaker can be placed inside the 2-L beaker to submerge the dialysis tubing in the ethanol solution.
Pour off ethanol and rinse the tubing 4 times with Milli-Q water, or until all EtOH has been removed.
Boil the tubing with EDTA (M.W. 292.24) and 10 mM sodium carbonate (M.W. 106) at pH 8.0 for 1 hour. In a 1.5-liter buffer, prepare 0.438 g EDTA and 0.159g sodium carbonate.
Rinse with Milli-Q water as step 4.
boil the tubing in Milli-Q water for 1 hr.
Tip off the final water, rinse, and replace with cold Milli-Q water containing 0.05% NaN3
Store at 4°C.
Prepare lipid stock
Prepare octyl-POE solution.
Solubilize the powder DMPC (from Avanti, 50mg per tube) to a final concentration of 2 mg/ml.
Aliquot the solution and store the lipids in the -20°C freezer.
Reconstitution
The mixture of protein-lipid-detergent (protein: 1-4 mg/mL, lipid: 10 mg/mL) before dialysis.
Dialyze against the buffer of HEPES (pH 7.0), 100 mM NaCl, 10 mM MgCl2, and 3 mM NaN3.
References
Rémigy, H.-W., Caujolle-Bert, D., Suda, K., Schenk, A., Chami, M., and Engel, A. Membrane protein reconstitution and crystallization by controlled dilution. FEBS Letters 555, 160-169 (2003).
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