cell lysis protocol

8/14/2019 Cell lysis protocol

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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, orinstrumental 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.
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Property SDS CHAPS Triton-X Tween 80

Charge anionic amphoteric non-ionic non-ionic

Denaturing yes no no no

Dialyzable yes yes no no

Ion-exchangable yes no no no

Strong Abs280 no no yes no

Interference Potential no no yes** yes*

Expensive no yes no no*Gel Filtration, native electrophoresis

**Gel Filtration, native electrophoresis, Lowry Assay

3. Passive lys is . 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, theconcentration 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.


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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.

Mechanical Techniques

1. Sonication. In this process, high frequency waves (20-50kHz) are generated electronically andtransmitted 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.

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

Kirb , Anthon J. The L soz me Mechanism Sorted After 50 ears. Nat. Struct. Biol.2001, 8, 737-739.


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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.


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Schematic of a homogenizer

cell lysis protocol

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