Cell disruption technology

Cell disruption technology

The cell disruption technique refers to a technique of destroying the cell membrane and the cell wall by external force, and releasing the cell contents including the target product component, and is the basis for separating and purifying the non-secretory biochemical substance (product) synthesized in the cell. Combined with significant advances in recombinant DNA technology and tissue culture technology, proteins previously thought to be difficult to obtain are now available for mass production.

Cell disruption resistance 　　

bacterial  　　

Almost all bacteria cell wall are peptidoglycan (peptidoglycan) composition, which is insoluble in the glycan chain (glycan chain), by means of a network structure of short peptides crosslinked, surrounds the cells, the cells having a certain Shape and strength. Short peptides generally consist of four or five amino acids, such as L -alanyl- D -glutamyl- L- lysyl- D- alanine. Moreover, D- amino acids and diaminopimelic acid are often present in short peptides . The main resistance of broken bacteria is the network structure of peptidoglycan. The density and strength of the network structure depend on the number of peptide bonds present on the glycan chain and the degree of cross-linking. If the degree of cross-linking is large, Then the network structure is dense.

Yeast  　　

The innermost layer of the yeast cell wall is composed of fine fibers of dextran, which constitutes a rigid skeleton of the cell wall, which makes the cells have a certain shape. A layer of glycoprotein is covered on the fine fiber, and the outermost layer is mannan. , covalently linked by a 1,6 -monophosphate diester bond to form a network structure. Inside the layer, there is a mannan - enzyme complex which can be covalently attached to the network or not. Like the bacterial cell wall, the resistance of the broken yeast cell wall is mainly determined by the tightness of the cross-linking of the wall structure and its thickness.

Fungus

There are mainly three kinds of polymers in the cell wall of mold, dextran (mainly linked by β-1,3 glycosidic bonds, some linked by β-1,6 glycosidic bonds), chitin (presented in the state of microfibers) and sugar protein. The outermost layer is a mixture of α- and β- glucan, the second layer is the network structure of glycoprotein, the glucan is combined with glycoprotein, the third layer is mainly protein, and the innermost layer is mainly chitin. Chitin microfibers are embedded in the protein structure. Like the cell walls of yeast and bacteria, the strength of the fungal cell wall is related to the network structure of the polymer. Not only that, but it also contains a fibrous structure of chitin or cellulose, so the strength is improved.

Plant cell

For the plant cell wall that has been grown, it can be divided into two parts: the primary wall and the secondary wall. The primary wall is formed during the growth phase of the cell. The secondary wall is a structure formed inside the primary wall after the cell stops growing. At present, the more popular primary cell wall structure is the " latitude and longitude " model proposed by Lampert et al . According to this model, the microfibrils of cellulose are applied layer by layer in the direction parallel to the plane of the cell wall, on the same level. The microfibrils are arranged in parallel, and the different directions are arranged in different directions, forming an independent angle to form an independent network, which constitutes the " jing " of the cell wall . The " latitude " in the model is a structural protein (rich in hydroxyproline). Protein, which is secreted by the cytoplasm, aligned perpendicular to the cell wall plane, and crosslinked by iso-dityrosine to form a structural protein network, and the radial microfibril mesh and the zonal structural protein network are cross-linked to each other. Form a more complex network system. Colloids such as hemicellulose and pectin are filled in the network, making the entire cell wall both rigid and elastic. In the secondary wall, the cellulose and hemicellulose content is much higher than the primary wall, the microfibrils of cellulose are arranged more tightly and regularly, and the deposition of lignin (polymer of the phenolic component) is present. Therefore, the formation of the secondary wall enhances the rigidity of the cell wall and gives the plant cells a high mechanical strength.

Cell disruption technology  　　

A variety of cell disruption methods have been developed to accommodate different uses and different types of cell wall disruption. The crushing method can be classified into two major categories: mechanical methods and non-mechanical methods.

Mechanical method  　　

- a high pressure homogenizer disruption, a high pressure homogenizer (homogenization) is a common device, which can be produced by a high-pressure positive displacement pump (positive displacenemt pump) and composition of the discharge valve (discharge valve), the discharge valve having a narrow aperture , its size can be adjusted. The cell slurry enters the pump body through the check valve, and under high pressure, it is forced to rush out in the small hole of the discharge valve and hit the impact ring. Due to the sudden decompression and high-speed impact, the cells are subjected to high liquid phase shearing. Broken by force. In the operation mode, a single pass through a homogenizer or a plurality of cycles can be used, or continuous operation can be performed. In order to control the temperature rise, the temperature can be adjusted with dry ice at the inlet to adjust the outlet temperature to about 20 °C . In industrial-scale cell disruption, multiple cycles of manipulation are often employed for cells that are difficult to break, such as yeast, and that have high concentrations or are in stationary phase of growth.

~ Skaking Bead: Place an equal volume of small tissue samples with high-density ZircoBeads in a sealable 2ml screw cap microtube , then add buffer and stabilizing components to a volume of 1.5ml , using 6500RPM high speed vertical vibration shaker 8 seconds, 8 seconds rest, and then vibration in 8 seconds. this method is the fastest method and can handle up to one sample. a machine can handle up to 2400 samples in one day. for small and A variety of people are very convenient . 　　

~ High-speed stirring bead grinding ( fine grinding ) Grinding is a commonly used method, which rapidly stirs the cell suspension with abrasives such as glass beads, quartz sand or alumina to break the cells. In industrial scale crushing, high speed bead mills are often used.

~ Ultrasonic fragmentation (ultrasonication) ultrasonic fragmentation ultrasonic probe ultrasonic oscillator emitted 15-25kHz treated cell suspension. There are different types of ultrasonic oscillators. The commonly used electroacoustic type is composed of a generator and a transducer. The generator can generate high-frequency current, and the transducer functions to convert electromagnetic oscillation into mechanical vibration. The ultrasonic oscillator can be divided into two types: the slot type and the probe directly inserted into the medium, and the latter is generally better than the former.

Non-mechanical method  　　

渗透 osmotic shock osmotic shock is a milder method of crushing cells placed in a high osmotic pressure solution (such as a certain concentration of glycerol or sucrose solution), due to osmotic pressure, cells The water will ooze out and the cells will shrink. When the equilibrium is reached, the medium is rapidly diluted, or the cells are transferred to water or buffer. Due to the sudden change of osmotic pressure, extracellular water rapidly infiltrates into the cells, causing The cells swell rapidly and rupture.

~ Freezing and thawing The cells are frozen at a low temperature (about -15 ° C ), then thawed at room temperature, and repeatedly broken to achieve wall breaking. Due to freezing, on the one hand, the hydrophobic bond structure of the cell membrane can be broken, thereby increasing the hydrophilic property of the cell, and on the other hand, the intracellular water crystallizes to form ice crystal grains, causing the cells to swell and rupture. This method can be used for cells with weak cell walls.

~ Enzymatic lysis The enzymatic hydrolysis is to treat the bacterial cells by the enzyme that dissolves the cell wall, and then partially or completely destroy the cell wall, and then destroy the cell membrane by osmotic pressure shock, etc., further increasing the permeability of the intracellular product. Sex. Lysozyme (Lysozyme) Gram-negative bacteria suitable for decomposing cells, when applied to gram-positive bacteria, supplemented with EDTA required to make it more efficiently on the cell wall. The cell wall of eukaryotic cells differs from prokaryotic cells in that different enzymes are required.

- chemical treatment Chemical treatment can dissolve cells or extract intracellular components. Chemical reagents such as acids, bases, surfactants and organic solvents

~ detergent crushing method ( detergents )

Protein renaturation

Using the density difference between the inclusion body and the cell debris, the inclusion body is separated from the cell debris and the soluble protein by centrifugation to obtain a clean inclusion body, and the inclusion body is refolded. In this way, a large amount of impurities such as impurities, nucleic acids, pyrogens and endotoxins are first removed, so that the subsequent separation and purification is simple. From this perspective, the formation of inclusion bodies is also beneficial for separation and purification.