Cell Lysis

Once a protein has been expressed in the host organism, the protein must be removed from the cellular enclosure to proceed in the purification process. This is accomplished by one of many approaches designed to disrupt the cellular membrane. In all cases, a suitable buffering system must be determined which stabilizes the protein of interest and is compatible with the method of choice. Several variables are important to consider when selecting a technique. 1. Volume - Some of the techniques listed below are very hard to scale up due to cost, time, or instrumental feasibility. 2. Type of cell - The outer membranes of some organisms is notoriously difficult to disrupt by chemical or enzymatic methods without compromising the structural integrity of the protein of interest. 3. Stability The mechanical techniques listed below all rely on an intense amount of energy or stress to rupture the cell membrane. This may compromise protein structure. 4. Purification process In some cases, the lysis method may influence downstream purification steps. This is particularly notable when working with some detergents. 5. Protein localization Some naturally secreted proteins may undergo the same process in the host organism, in which case the protein will NOT be in the cytosol. Instead, it will be trapped in the periplasm or completely secreted to the growth media.

Equipment Independent Techniques

1. Freeze-thaw. In this approach, the cells are suspended in an appropriate experimental buffer and subjected to repeated freezing and thawing. Enzymatic lysis (see below) may be used to improve this method. Advantages: No specialized equipment is necessary. Disadvantages: Very slow.

2. Chemical lysis. Resuspending the cells in a detergent containing media will dissolve the outer membrane and allow cytosolic proteins to be extracted. Some detergents are very deleterious to protein structure while others are hard to remove and will influence subsequent purification steps. Therefore, choice of detergent is particularly important. There a number of commercially available optimized detergent containing solutions for this technique (e.g. B-PER from Pierce). Table 1 lists some common detergents and strengths/weaknesses. Advantages: Fast and cheap. Disadvantages: May influence protein structure or purification.

Property Charge Denaturing Dialyzable Ion-exchangable Strong Abs280 Interference Potential Expensive

SDS anionic yes yes yes no no no

CHAPS amphoteric no yes no no no yes

Triton-X non-ionic no no no yes yes** no

Tween 80 non-ionic no no no no yes* no

*Gel Filtration, native electrophoresis **Gel Filtration, native electrophoresis, Lowry Assay

3. Passive lysis. For easily disrupted cells, it may be possible to take advantage of the high intracellular concentration of solute molecules to disrupt the cellular membrane. By suspending the cells in a low ionic strength buffer, hypotonic conditions are produced and the cells will swell and burst due to osmotic pressures. For most bacteria, the presence of a cell wall necessitates the use of an enzymatic approach in addition to an osmotic gradient. Advantages: Cheap Disadvantages: Not universally applicable.

The passive diffusion of water molecules through cell membranes, indicated by arrows in the figure above, is facilitated by a family of proteins known as aquaporins. Under hypotonic osmotic conditions, the concentration of electrolytes (ovals) is higher on the intracellular side of the membrane resulting in the uptake of water leading to swelling and rupturing of the cell membrane.

4. Enzymatic Lysis. Although not physically able to destroy the cell membrane, enzymatic degradation of the cell wall is well established and widely used. If the protein of interest is secreted and trapped in the periplasm, enzymatic lysis is all that is necessary. However, if the protein is localized within the protoplast (cells without the cell wall), an additional method must be used in tandem with this approach. Enzymatic and passive lysis are often used together. Although other enzymes are available, lysozyme is the most commonly used. Advantages: No specialized equipment necessary. Disadvantages: Not always reproducible. Enzyme Stability can be an issue. Can be expensive to scale up.

Mechanism of lysozyme hydrolysis of -N-acetylmuramic acid (NAM) (14) N-acetylglucosamine (NAG) glycosidic linkage. Green indicate residues from lysozyme. Red show bonds that are formed during the intermediate. Note that only one of these bonds is actually formed. See reference for details
Kirby, Anthony J. The Lysozyme Mechanism Sorted After 50 years. Nat. Struct. Biol. 2001, 8, 737-739.

Mechanical Techniques
1. Sonication. In this process, high frequency waves (20-50kHz) are generated electronically and transmitted mechanically to a suspension of cells by immersing a metal probe that is vibrating at the ultrasonic frequency into the suspension of cells. This process produces small cavities of low pressure around the cells, ultimately resulting in cell lysis. This process generates a significant amount of heat which can lead to protein unfolding. Short (5-15 sec) sonication burst cycles are often used with longer intermittent periods to allow the solution to cool. Advantages: Useful for most cell types. Easily applicable to large or small scale. Disadvantages: Requires specialized equipment. Loud - hearing protection required.

2. Cell Bomb. In a cell bomb, suspended cells are placed in a fixed volume vessel and pressurized (typically with N2 gas) to ~25,000 psi. This process results in large amounts of dissolved gases. The pressure is rapidly released causing the dissolved gases to quickly disperse, forming bubbles that ultimately destroy the membrane. Advantages: Fast. Inexpensive equipment requirement. Disadvantages: Not applicable to all cell types.

3. Homogenizer (French Press). This method utilizes sheer force as a method to disrupt cell membranes. The sample is fed through a chamber with an exit port smaller than the diameter of a cell. The hydraulic pressure is great enough to force the cells through the exit port, resulting in the membrane bursting and releasing intracellular components. Advantages: Fast and ideal for large scale lysis. Useful for most cell types. Disadvantages: Expensive equipment required. Prone to clogs.

Schematic of a homogenizer