Protein Characteristics and Protein Separation Technology

Proteins generally exist as complex mixtures in tissues or cells, and each type of cell contains thousands of different proteins. The separation and purification of proteins is an arduous and arduous task. So far, there is no single or a set of ready-made methods that can extract any kind of protein from a complex mixture, but for any kind of protein It is possible to choose a set of appropriate separation and purification procedures to obtain high-purity products.

The overall goal of protein purification is to try to increase the purity or specific activity of the product. The requirements for purification are reasonable efficiency, speed, yield and purity, and separate the required protein from all other components of the cell, especially unwanted contaminants. , While still retaining the biological activity and chemical integrity of this peptide.

The reason why a protein can be purified from thousands of protein mixtures is that different proteins have very different physical, chemical, physicochemical and biological properties. These properties are due to the amino acid of the protein. Due to the difference in sequence and number, the amino acid residues attached to the polypeptide backbone can be positively charged, negatively charged, polar or non-polar, hydrophilic or hydrophobic. In addition, the polypeptide can be folded into a very definite Secondary structure (α helix, β sheet and various turns), tertiary structure and quaternary structure, forming a unique size, shape and distribution of residues on the protein surface, using the difference between the protein to be separated and other proteins The difference in nature can design a set of reasonable fractionation steps.

The protein mixture can be separated according to the method corresponding to the different properties of the protein:

1. Molecular size
   Different kinds of proteins have certain differences in molecular size, and some simple methods can be used to make the protein mixture get preliminary separation.
   1.1 Dialysis and ultrafiltration
   Dialysis is very commonly used in purification to remove salts (desalting and replacement buffers), organic solvents, and low molecular weight inhibitors. The molecular weight cut-off of the dialysis membrane is about 5000. For example, the enzyme solution with a molecular weight less than 10000 may leak. It is very commonly used in purification to remove salts, organic solvents, and low molecular weight inhibitors. Ultrafiltration is generally used for concentration and decolorization
   1.2 Centrifugal separation of replacement buffer
   Many enzymes are enriched in a certain organelle. After homogenization, a certain subcellular component is obtained by centrifugation, which enriches the enzyme by 10-20 times, and then purifies the specific enzyme. Differential centrifugation has low resolution and is only suitable for rough extraction or concentration. Rate zone method, if the centrifugation time is too long, all the substances will be precipitated, so it is necessary to select the best separation time to obtain fairly pure subcellular components for further purification, avoiding the precipitation of large and small components together in differential centrifugation Problem, but the capacity is small and can only be used for small amounts of preparation. Commonly used separation media for isocratic gradient centrifugation are sucrose, polysucrose, cesium chloride, potassium bromide, sodium iodide, etc.
   1.3 Gel filtration
   This is one of the most effective methods for separating protein mixtures based on their molecular size. Take care to make the molecular weight of the protein to be separated fall within the working range of the gel. Choosing different molecular weight gels can be used for desalting, replacing buffers, and using molecular weight differences to remove heat sources.

2. shape
   When the protein moves through the solution by centrifugation, or when it moves through the membrane, gel filter filler particles, or the small holes in the electrophoresis gel, it will be affected by the shape: for two proteins of the same quality, the globular protein has a smaller size. Effective radius (Stoke’s radius), the friction force encountered when passing through the solution is small, the sedimentation is faster and appears larger than other shapes of proteins; on the contrary, in size exclusion chromatography, the spherical protein with a smaller Stoke radius It is easier to diffuse into the inside of the gel filtration packing particles and elute later, so it appears smaller than other shapes of protein.

3. Solubility
   Use the difference of protein solubility to distinguish the commonly used methods of various proteins. There are many external factors that affect protein solubility, among which are: pH, ionic strength, dielectric constant and temperature of the solution. However, under the same specific external conditions, different proteins have different solubility. Appropriately change the external conditions to control the solubility of a certain component in the protein mixture
   3.1 pH control and isoelectric point precipitation
   Proteins are generally less soluble at their isoelectric point.
   3.2 Salting and salting out of protein
   3.3 Classification of organic solvents
   The solubility of protein in different solvents is very different, ranging from basically insoluble (<10μg to="" extremely="" soluble="">300mg/ml). Variable factors that affect protein solubility include temperature, pH, solvent polarity, ionic properties, and ionic strength. The concentration of the organic solvent that causes protein precipitation is different, so the concentration of the organic solvent can be controlled to separate the protein.
   Water-soluble non-ionic polymers such as polyethylene glycol can also cause protein precipitation.
   3.4 Temperature
   Different proteins have different solubility and activity at different temperatures. Most proteins are relatively stable at low temperatures, so the separation operation is generally carried out at 0°C or lower.

4. Charge
   The net charge of a protein depends on the sum of the positive and negative charges carried by the amino acid residues. For example, a neutral solution with a net negative charge is called an acidic protein.
   4.1 Electrophoresis
   It is not only an important means for separating protein mixtures and identifying protein purity, but also a very useful method for studying protein properties.
   The resolution of isoelectric focusing is very high, and the difference of pI can be separated by 0.02pH.
   The resolution of 2D-PAGE separation of proteins has been developed to 100,000 protein spots.
   4.2 Ion exchange chromatography
   Change the salt ionic strength, pH, and (anion, cation) ion exchange packing in the protein mixture solution. Different proteins have different adsorption capacities for different ion exchange packings. Proteins are separated due to different adsorption capacities or non-adsorption.
   Elution can be done by keeping the eluent composition constant, or by changing the salinity or pH of the eluent. The latter can be divided into segmented elution and gradient elution. Gradient elution generally has good effect and high resolution, especially when using ion exchangers with small exchange capacity and sensitive to salt concentration, gradient elution is often used. By controlling the volume of the eluent (compared to the volume of the column bed), the salt concentration and the pH, the sample components can be eluted from the ion exchange column separately.
   The type and number of side chain groups exposed on the outer surface of protein molecules are different, so the charge of the buffer at a certain pH value and ionic strength is different.

5. Charge distribution
   The charged amino acid residues can be evenly distributed on the surface of the protein, and can be combined with the cation exchange column with appropriate strength or with the anion. Because most proteins cannot be combined with two types at the same time under a single solvent condition. The ion exchange column is combined with the ion exchange column, so it can be purified by this property; the charged amino acid residues can also be distributed in clusters, so that one area has a strong positive charge and another area has a strong negative charge, which is strongly acidic or strong. It can only be combined with cation exchange resin or anion exchange resin at extreme pH. For example, calmodulin can only be combined with cation exchange resin at pH 2.

6. Hydrophobicity
   Most of the hydrophobic amino acid residues are hidden inside the protein, but some are on the surface. The number and spatial distribution of the hydrophobic amino acid residues on the protein surface determine whether the protein has the ability to combine with the hydrophobic column packing to use it for separation.
   Because it is cheap and the purified protein has biological activity, it is a universal tool for separating and purifying protein. The protein in the high-concentration saline solution is retained on the column, and the protein is eluted from the column in the low-salt or aqueous solution, so it is especially suitable for the mother liquor after precipitation and separation of the concentrated ammonium sulfate solution and the target product after the precipitation is dissolved by the salt. The solution is directly injected onto the column, and of course, the therapeutic protein extract of E. coli with 7 mol/guanidine hydrochloride or 8 mol/L urea is also directly injected onto the column, and the renaturation is carried out at the same time as the separation.

7. Density
   The density of most proteins is between 1.3~1.4g/cm3. This property is generally not used in protein fractionation. However, the density of proteins containing a large amount of phosphate or lipids is significantly different from that of general proteins. Density gradient centrifugation can be used with Most of the protein is separated.

8. Purified markers constructed by genetic engineering
   By changing the cDNA to add a few extra amino acids at the amino or carboxyl terminus of the expressed protein, this added tag can be used as an effective basis for purification.
   8.1 GST fusion vector
   Make the protein to be expressed and glutathione S transferase expressed together, then use Glutathione Sepharose 4B for affinity purification, and then use thrombin or factor Xa to cut.
   8.2 Protein A fusion vector
   The protein to be expressed and the IgG binding site of protein A are fused together for expression, and purified with IgG Sepharose.
   8.3 Histidine-tagged Chelating Sepharose
   One of the most common markers is to add 6-10 histidines to the amino terminus of the protein. Under normal or denaturing conditions (such as 8M urea), with the help of its ability to bind tightly to the Ni2+ chelating column, imidazole is used. Elute or lower the pH to 5.9 to fully protonate histidine and no longer bind with Ni2+ to make it purified.
   Recombinant protein has been integrated into the purification concept when designing and constructing. The sample is mostly mixed with broken cells or soluble products. The expanded bed adsorption technology STREAMLINE is suitable for coarse separation.

9. Affinity
   Combines the characteristics of high efficiency and fast separation speed. Ligands can be enzyme substrates, inhibitors, cofactors, specific antibodies,
   After adsorption, the method of changing the ionic strength and pH of the buffer can be taken off by listening to your ears. It can also be eluted with a higher concentration of the same ligand solution or a ligand solution with stronger affinity
   The ligands of the affinity chromatography stationary phase and the biomolecules are separated from each other by the different affinity of the special biological macromolecules. According to the level of affinity selectivity, they are divided into: group affinity chromatography, stationary phase The ligands have a strong affinity for a class of groups. For example, a class of proteins or glycoproteins containing glycosyls show particularly strong adsorption capacity for triazine dyes; high-selectivity (specific) affinity chromatography, ligands only have a particularly strong affinity for a certain protein . Such as the specific adsorption of monoclonal antibodies to antigens.
   In addition to specific adsorption, affinity chromatography will still adsorb some impurity proteins due to molecular misidentification and non-selective forces between molecules. In addition, the ligands in the elution process will inevitably fall off into the separation system.
   Combined with ultrafiltration, the advantages of the two are concentrated to form ultrafiltration affinity purification, which has the advantages of high separation efficiency and large-scale industrialization, and is suitable for initial separation.
   According to the different ligands, it can be divided into:
   (1) Metal chelating medium
   The transition metal ions Cu2+, Zn2+ and Ni 2+ are bonded to the phase phasing in the form of imine complexes, because these metal ions form coordination bonds with tryptophan, histidine and cysteine , Thus forming the imine metal-protein chelate, so that the protein containing these amino acids is adsorbed by the stationary phase of this metal chelate affinity chromatography. The stability of the chelate is controlled by the dissociation constant of a single histidine and cysteine, which is also affected by the pH and temperature of the mobile phase. The controlled conditions can separate different proteins from each other.
   (2) Small ligand affinity medium
   Ligands are arginine, benzamide, calmodulin, gelatin, heparin, lysine and so on.
   (3) Antibody affinity medium
   That is, immunoaffinity chromatography, the ligands are recombinant protein A and recombinant protein G, but protein A is more specific than protein G, and protein G can bind more IgG from different sources.
   (4) Pigment affinity medium
   The effect of dye chromatography mainly depends on the affinity of the dye ligand and the enzyme, but also the type of elution buffer, ionic strength, pH value and the purity of the sample to be separated. There are two kinds of ligands: Cibacron Blue and Procion Red. Under certain conditions, the immobilized dye can act as a cation exchanger. In order to avoid this phenomenon, it is best to operate when the ionic strength is less than 0.1 and the pH is greater than 7.
   (5) Affinity medium of exogenous lectin
   Ligands include concanavalin, lentil lectin and malt lectin. The solid-phase lectin can react reversibly with several carbohydrate residues and is suitable for the purification of polysaccharides and glycoproteins.

10. Forces between non-polar groups
    The interaction force (non-selective dispersion force or London force) between the non-polar group in the solute molecule and the non-polar stationary phase is related to the polar group of the solute molecule and the polar molecule in the mobile phase in the opposite direction. The difference in force is separated. Because the displacer in the mobile phase is an organic solvent with less polarity than water (such as methanol, acetonitrile, tetrahydrofuran, etc.), these organic solvents may irreversibly denature many protein molecules; an ion pair reagent (such as three The presence of fluoroacetic acid, formic acid, phosphoric acid, etc.) can enable the separation to proceed effectively and obtain high mass recovery; the separation must be carried out in an acidic medium (generally pH is between 2 and 3), and some proteins will be in the latter two. Under these conditions, irreversible molecular conformation changes are produced, so the separation and purification of biological macromolecules is limited, but it is an effective method for proteins whose molecular conformation changes are reversible.
    Normal phase chromatography has relatively few applications in the separation and purification of biological macromolecules, because the solvents used are very expensive.

11. Reversible association
    Under certain solution conditions, some enzymes can polymerize into dimers, tetramers, etc., while under another condition, they form monomers. For example, under these two different conditions, they can be classified according to size. Separate.

12. Stability
    12.1 Thermal stability
    Most proteins will unfold or precipitate when heated to 95°C. Using this property, a protein that retains its soluble activity after such heating can be easily separated from most other cellular proteins.
    12.2 Stability of proteolysis
    Treat the supernatant with protease to digest the contaminated protein, leaving behind the protein resistant to proteolysis.

13. Partition coefficient
    That is, the use of aqueous two-phase extraction and separation, the commonly used biological material separation systems are: polyethylene glycol (PEG)/dextran (DEXTRAN), PEG/phosphate, PEG/ammonium sulfate, etc. Due to its high water content, the non-toxicity of selected polymers and salts to enzymes, and the common use of separation equipment with the chemical industry, it has attracted more attention in the industry.
    The distribution behavior is affected by factors such as polymer molecule size, phase concentration, PH, inorganic salt type, etc.
    Development: New aqueous two-phase separation technologies such as affinity two-phase extraction and membrane separation two-phase extraction.
    Protein purification in bidirectional aqueous system
    The incompatibility of some polymers mixed with water leads to a two-phase system of liquid-liquid distribution technology that depends on the concentration of the polymer. It can consist of two different highly water-soluble polymers or one polymer each. Salt is used to remove branched cell debris from the microbial homogenate and further purify it in subsequent distribution steps. The selectivity of separation generally increases with the size of the separated molecules or particles.

14. Surface activity
    14.1 Foam separation
    The protein solution has surface activity, the gas is bubbled in the solution, and the bubbles are separated from the main body of the liquid phase and are enriched at the top of the tower to achieve the purpose of separation and concentration.
    14.2 Reverse micelle phase transfer method
    The reverse micelle phase transfer method is a new type of separation technology that emerged in the 1980s. It uses reverse micelles (reverse micelles) spontaneously formed by surfactant molecules in organic solvents to increase water-soluble protein molecules under certain conditions. Dissolve into the polar nucleus (pool) of the reverse micelle, and then create conditions to extract the protein to another water phase, realize the phase transfer of the protein, and achieve the purpose of separating and purifying the protein. The protein molecules in the reverse micelles are protected by the surrounding water molecules and the polar heads of surfactants, and still maintain a certain degree of activity, even showing super activity. Since protein solubilization in reverse micelles is related to the electrostatic interaction between the charges on the protein and the internal surface charges of the reverse micelles, and the size of the reverse micelles, the types of surfactants, the pH value of the aqueous solution, and ionic strength are all affected. Phase transfer of reverse micelles to proteins. It is reported that AOT/iso-octane reverse micelles are used for phase transfer of yeast lipase.
    14.3 The polymer-salt-water-liquid-solid benzene extraction system is a new extraction system developed in the country in the 1990s. It has been successfully used for the extraction of metal ions and strong adsorption of biologically active substances such as Bentholic acid dehydrogenase. Defects such as emulsification and easy phase formation. After phase formation, the liquid phase can be directly poured out to separate the liquid and solid phases. It does not require special technical treatment, does not require organic solvents, and is non-toxic. Stable and protective effect, good extraction and separation selectivity, low consumption, easy to scale up, a new technology for separating bioactive substances, covalently in the polyethylene glycol/dextran aqueous two-phase extraction system of modified polyethylene glycol Function: Mainly used for the purification of sulfhydryl-containing enzymes. It is covalently bonded to the chromatographic medium. The coupling is reversible. The low molecular compound that can reduce the disulfide bond is eluted. When the buffer is adsorbed, if it already contains disulfide bonds, first open it with a reducing agent.

Related Articles:

Protein Isolation and Purification Techniques

Protein Purification Service Company