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.
Membrane preparation should be performed immediately after cell disruption.
All steps are carried out at 4 °C or on ice.
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.