Cell Disruption and Membrane Preparation

Methods for cell disruption are host dependent and essentially the same protocols are used for recovery of membrane proteins as for water-soluble proteins. Cell disruption yields a suspension of membrane fragments/vesicles that contains the membrane proteins. The suspension also contains soluble proteins, remaining intact cells, various cell debris, and other material as contaminants. These contaminants may need to be removed, depending on the purification procedure used. Differential centrifugation is the standard approach for the isolation of membrane fragments/vesicles after cell disruption.

The pellet from cell harvest is resuspended in a suitable buffer for cell disruption (e.g., PBS). DNase is added to reduce viscosity. It is useful to add a protease inhibitor cocktail to reduce possible protein degradation. A selection of commonly used techniques for cell disruption are summarized in Table 1.2.

Cell Disruption from Frozen E. coli Cell Paste with Overexpressed Membrane Protein

Cell Disruption

1. For each gram of cell paste combine, from the above solutions, 5 mL PBS, 5 µL MgCl₂, 50 µL Pefabloc, 5 µL DNase, and 80 µL lysozyme. Mix until the suspension is homogenous.
2. Sonicate on ice. Use the manufacturer’s recommended settings for amplitude and time for the probe being used (e.g., 5 min of accumulated time; 9 s on, 5 s off).
3. Continue immediately with the membrane preparation.

Membrane preparation should be performed immediately after cell disruption.

Membrane Preparation from E. coli.

All steps are carried out at 4 °C or on ice.

1. Centrifuge at 24 000 × g for 12 min. Collect the supernatant.
2. Centrifuge the supernatant at 150 000 × g for 45 min. Remove the supernatant and resuspend the pellet in 10 mL PBS. The pellet contains the membrane fraction.
3. Centrifuge the resuspended pellet at 150 000 × g for 45 min. Remove the supernatant and resuspend the pellet in 5 mL PBS.
4. Determine protein concentration using standard methods for soluble proteins such as the Biuret method or the bicinchoninic acid (BCA) method.
5. For storage, rapidly freeze the membrane suspension dropwise using liquid nitrogen and store at –80 °C.

Different cell disruption protocols may give rise to different size fragments; the centrifugation speed needs to be optimized accordingly.

Water crystals formed upon slow freezing may harm membrane proteins. Fast freezing by submersion of the membrane suspension in liquid nitrogen forms amorphous ice structures thus reducing the negative effects of freezing (some researchers avoid freezing completely and always perform membrane protein preparation from cell to pure protein as fast as possible, without interruptions; see next point).

For unstable membrane proteins, it may be beneficial to proceed directly with purification after the preparation of membranes, and thus avoid freezing and storing the membranes.

For small scale (0.5 to 50 mL) membrane preparations from E. coli, a procedure with lysozyme treatment followed by osmotic shock and centrifugation is often efficient.

To facilitate protein purification, it can be useful to separate the inner and outer membranes from E. coli membrane preparations. This can be particularly helpful for large (1 to >10 L) preparations. The inner membrane can be selectively solubilized with 2% N-lauroylsarcosine. The outer membranes can then be recovered in the pellet after a 1 h centrifugation. An alternative is to separate the inner and outer membranes by a long (~10 h) sucrose gradient centrifugation, following cell disruption.

It is sometimes possible to omit the fairly lengthy and cumbersome membrane preparation step. The alternative is to first disrupt the cells and then directly solubilize membrane proteins by the addition of detergent to the cell lysate, with no prior isolation of membranes. The resulting “solubilisate” can be used for chromatography directly. By using chromatography columns that accept direct loading of unclarified homogenized cell lysate and detergent-treated unclarified lysate (e.g., HisTrapTM FF crude columns), histidine-tagged membrane proteins can be purified directly from the cell lysate.