Protein Separation And Purification

At the end of the research, it depends on the expression product of the group. Whether it is used for detection or for cotton protection, the expressed protein needs to be separated and purified. However, the properties of the protein are different, so the method is different.

The primary, secondary, tertiary, and quaternary structure of a protein determine its physical, chemical, biochemical, physical, chemical, and biological properties.In addition, it summarizes the differences in the properties of different proteins or changes the conditions to make them different. Using a variety of properties at the same time, in the case of both yield and purity, choose the method of protein purification.

Proteins generally exist in the form of complex mixtures in tissues or cells, and each type of cytotoxicity contains thousands of different proteins. The separation and purification of protein is an arduous task. The overall goal of the protein purification is trying to increase the purity of products or specific activity, requires reasonable, speed, high yield, purity for purification. And seperate the protein from cell of other all components, especially unwanted impure protein, while still retaining the biological activity and chemical integrity of the 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 caused by different sequences and number of proteins’ amino acids, and connected in the main chain of the polypeptide amino acid residues is positive, burden on electricity, polar or non-polar, hydrophilic or hydrophobic.

In addition, the polypeptide can fold into a very determine secondary structure of (alpha helix, beta, folding and various Angle), tertiary structure, and quaternary structure. Formed unique size, shape, and distribution of residues on the protein surface and a set of reasonable fractional separation steps can be designed by taking advantage of the difference in properties between the protein to be separated and other proteins.

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

1 Molecular Size

Different types 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 and can 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 of less than 10000 may leak. 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, so that the enzyme is enriched by 10-20 times, and then the specific enzyme is purified. Differential centrifugation, low resolution, only suitable for rough extraction or concentration.

Rate zonal, if the centrifugation time is too long, all the substances will be precipitated. Therefore, 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 in differential centrifugation. But the capacity is small and can only be used for small quantities.

Commonly used media for density gradient centrifugation include sucrose, polysucrose, cesium chloride, potassium bromide, and sodium iodide.

1.3 Gel Filtration

This is one of the most effective methods for separating protein mixtures based on their molecular size. Take care that the molecular weight of the protein to be separated falls 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

Proteins are affected by their shape when they move through the solution during centrifugation, or when they move through membranes, gel filtration filler particles, or small holes in electrophoresis gels.

For two proteins of the same mass, the cyclic protein has a smaller effective radius (Stokes radius). The friction encountered when settling through the solution is small, and the settling is faster and appears larger than other shapes of protein. On the contrary In size exclusion chromatography, globular proteins with a smaller Stoke radius are more likely to diffuse into the inside of the gel filtration packing particles and elute later, so they appear smaller than other shapes of proteins.

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 the main ones are: solution pH, ionic strength, dielectric constant and temperature. But 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 Organic Solvent Classification Method

The solubility of protein in different solvents is very different, ranging from basically insoluble (<10μg/ml) 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 the protein precipitation is different, so the concentration of the organic solvent can be controlled to separate the protein.

Water-soluble non-ionic polymers (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 method for separating protein mixtures and identifying protein purity, but also a very useful method for studying protein properties.

The isoelectric focusing resolution is very high, and the pI can be separated with a difference of 0.02pH.

The resolution of 2D-PAGE separation of proteins has been developed to 100,000 protein spots.

4.2 Ion Exchange Chromatography

Changing 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, and proteins are separated due to different adsorption capacities or not being adsorbed.

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 the use of ion exchangers with small exchange capacity and sensitive to salt concentration. Control the volume of the eluent (compared to the volume of the column bed), salt concentration and pH, and sample The components can be eluted separately from the ion exchange column.

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 uniformly distributed on the surface of the protein and can be combined with the cation exchange column with appropriate strength or with the anion. Since most proteins cannot bind to both types of ion-exchange columns in a single solvent, they can be purified with 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 be combined with cation exchange resin or cation 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 for separation.

It is cheap and the purified protein has biological activity, it is a universal tool for separating and purifying the protein.

The protein in the high-concentration saline solution is retained on the column, and eluted from the column in low-salt or aqueous solution. Therefore, it is especially suitable for the mother liquor after precipitation and separation of concentrated ammonium sulfate solution, and the solution containing target products after the precipitation is dissolved with salt to be directly injected into the column.

7mol/ guanidine hydrochloride or 8mol/L urea is also suitable for the treatment of Escherichia coli protein extract directly injected into the column, and the separation was also performed at the same time of renaturation.

7 Density

The density of most proteins is between 1.3~1.4g/cm3. This property is generally not commonly used in protein fractionation.

However, the density of proteins containing a large amount of phosphate or lipid is obviously different from that of common proteins, so most of the proteins can be separated by density gradient centrifugation.

8 Purification Marker Constructed By Genetic Engineering

By changing the cDNA to add few extra amino acids at the amino-terminal or carboxyl-terminal of the expressed protein, it can be used as an effective basis for purification.

8.1 GST Fusion Vector

The protein to be expressed is expressed together with glutathione S-transferase, and then purified with Glutathione Sepharose 4B, and then cut with thrombin or factor Xa.

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 labels is to add 6~10 histidines to the amino end of the protein. Under normal or denaturing conditions (8M urea), with the help of its ability to bind tightly to the Ni2+ chelating column, wash with imidazole Removal or lowering the pH to 5.9 makes histidine fully protonated and no longer binds to 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 rough separation.

9 Affinity

Combines the characteristics of high efficiency and fast separation speed. Ligands can be enzyme substrates, inhibitors, cofactors, and specific antibodies.

After adsorption, the ionic strength and pH of the buffer can be changed to elute the target protein. It can also be eluted with a higher concentration of the same ligand solution or a ligand solution with a stronger affinity.

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 in the form of imine complexes. Since these metal ions form coordinate bonds with tryptophan, histidine, and cysteine, Thereby forming 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 control conditions can separate different proteins from each other.

(2) Small Ligand Affinity Medium

Ligands include arginine, benzamide, calmodulin, gelatin, heparin and lysine.

(3) Antibody Affinity Medium

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 dye ligand and magnitude of their affinity with enzyme. It is also related to 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, lentils 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 displacing agent in the mobile phase is an organic solvent with less polarity than water, these organic solvents may cause irreversible denaturation of many protein molecules. The ion pair reagent must be present in the mobile phase to make the separation effective and obtain high Mass recovery rate. The separation must be carried out in an acidic medium, and some proteins will produce irreversible molecular conformation changes under the latter two conditions, so the separation and purification of humans in biological macromolecules is limited.

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.

12 Stability

12.1 Thermal Stability

Most proteins will unfolding 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 resistance proteins to proteolysis.

13 Distribution Coefficient

Using aqueous two-phase extraction and separation, commonly used biological material separation systems are: polyethylene glycol (PEG)/dextran (DEXTRAN), PEG/phosphate, PEG/ammonium sulfate, etc. Owing to its high water-content ratio, the selected polymer and salt are non-toxic to enzymes. And separation equipment is also used in the chemical industry, it is paid attention to in the industrial.

Development: new dual-aqueous phase separation technology with affinity dual-aqueous phase extraction and membrane separation dual-aqueous phase extraction, etc.

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 80s. It uses the reverse micelles spontaneously formed by surfactant molecules in organic solvents. Under certain conditions, the water-soluble protein molecules are solubilized into the reverse micelles. In the polar nucleus, the conditions are created to extract the protein to another water phase to 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. It is reported that AOT/iso-octane reversed micelles are used for phase transfer of yeast lipase.

Related Reading:

Intact Protein Analysis

Proteomics Service

Protein Precipitation Technical Guide

Plasma Protein Binding Assay