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.

Table 1.2 Overview of techniques for cell disruption to yield a suspension of membrane vesicles

Technique	Principle	Advantages (+) / Disadvantages (-)
Liquid shear pressure (e.g., French press)	Rapid pressure drop by transferring the sample from a chamber at high pressure through an orifice into a chamber at low pressure	+ Fast and efficient, also for large volumes - Causes heating of the sample (cooling is   required)
Ultrasonication	Cells disrupted by high frequency sound	+ Simple - Causes heating of the sample, which        can be difficult to control by cooling - Proteins may be destroyed by shearing - Noisy - Not for large volumes
Glass bead milling	Agitation of the cells with fine glass beads	+ Useful for cells that are more difficult to   disrupt (e.g., yeast) - Somewhat slow and noisy
Osmotic shock	Change from high to low osmotic medium	+ Simple, inexpensive - Only useful for disruption of cells with   less robust walls (e.g., animal cells)
Repeated freezing and thawing	Cells disrupted by repeated formation of ice crystals; usually combined with enzymatic lysis	+ Simple, inexpensive + Yields large membrane fragments - Slow - May damage sensitive proteins and dissociate membrane protein complexes - Low yield
Enzymatic lysis	Often used in combination with other techniques, e.g., freezethawing or osmotic shock; lysozyme is commonly used to break cell walls of bacteria	+ Gentle + Yields large membrane fragments - Slow - Low yield

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

Solutions

PBS:	10 mM phosphate, 2.7 mM KCl, 137 mM NaCl, pH 7.4
MgCl2:	1 M
PefablocTM:	100 mM
DNase:	20 mg/mL
Lysozyme:	10 mg/mL

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.