How to purify protein with liquid chromatography methods?

General Principle For Protein Purification

Author: Ning Mu Ph. D., Guowei Sun                 Translator: Tian Jing 


The physical, chemical, and biological properties of different proteins are the target protein purification principle from tens of thousands of kinds of the mixture. These properties are caused by the different numbers of the sequence of amino acids and proteins. The amino acid residue which connects in the main chain of the polypeptide amino acid residues is positive, negative, polar or non-polar, hydrophilic or hydrophobic. And peptides can fold into a very determine the secondary structure of (α helix, β folding, and various angles), tertiary structure, and quaternary structure, which formed a unique size, shape, and distribution on the surface of the residues in the protein. A set of reasonable separation steps is designed by taking advantage of the property differences between proteins. The protein mixture can separate according to different properties of protein and corresponding methods.

The main methods for protein purification

Ion exchange, affinity, and reverse-phase are the most popular methods for protein purification. Here are some main principles for protein purification in common technologies.

protein purification in hplc system

1. Molecular Size

Different kinds of proteins have some differences in molecular size. Some simple methods can be used to separate the protein mixture.

1.1 Dialysis and Ultrafiltration

Dialysis is commonly used in purification to remove salts (desalination and replacement buffers), organic solvents, low molecular weight inhibitors, etc. Ultrafiltration is commonly used to concentrate and displace solutions.

1.2 Centrifuge the displacement buffer

Many enzymes were enriched in a certain organelle. After homogenizing and centrifuging, a certain subcellular component is obtained, which enriched the enzyme by 10-20 times. Then purify the specific enzyme. Differential centrifugation, low resolution, only suitable for crude extraction or concentration. For example, if the centrifugation time is too long, all substances will be precipitated, so the optimal separation time should be selected to obtain pure subcellular components for further purification. This method avoids the problem of precipitation of components of different sizes from the heart, but it has a small capacity and can only be used for a small amount of preparation. Sucrose, sucrose polysaccharide, cesium chloride, potassium bromide, and sodium iodide are commonly used in centrifugation with an equal density gradient.

1.3 Gel Filtration (GF)

This is one of the most efficient ways to separate protein mixtures according to their molecular size. Making sure that the molecular weight of the target protein falls within the working range of the gel. Different molecular weight gels can be used for desalting, displacing buffer, and removing heat sources by molecular weight difference.

2. Shape

The shape of the protein effect as it moves through the solution by centrifugation, or through the pores in the membrane, gel filter filler particles, or electrophoretic gels. For two proteins of the same mass, the globular protein has a smaller effective radius (stoker radius), which causes less friction when it sinks through the solution. In contrast, globular proteins with a smaller stoker radius are more likely to diffuse into the gel filter filler particles and elute later, thus appearing smaller than proteins of other shapes.

3. Solubility

A method commonly used to separate proteins by using their different solubility. There are many external factors that affect the solubility of proteins, including solution pH, ionic strength, dielectric constant, and temperature. However, under the same specific external conditions, different proteins have different solubilities. The solubility of a component in the protein mixture is controlled by changing the external conditions appropriately.

3.1 pH control and isoelectric precipitation

Proteins are generally less soluble at their isoelectric points.

3.2 Organic solvent separation method

The solubility of proteins in different solvents varies greatly from basically insoluble (<10ug/ml) to easily soluble (>300mg/ml). The concentration of organic solvent causing protein precipitation is different, so controlling the concentration of organic solvent can separate protein. Water-soluble nonionic polymers such as polyethylene glycol can also precipitate proteins.

3.3 Temperature

Different proteins have different solubility and activity at different temperatures. Most proteins are stable at low temperatures, so the separation process is usually performed at 0℃ 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.

4.1 Electrophoresis

It is not only an important method for separating protein mixtures and identifying protein purity, but also a useful method for studying protein properties. Isoelectric focusing resolution is very high, pI can be separated with a difference of 0.02pH.2D-PAGE protein isolation resolution has been developed to 100,000 protein points.

4.2 Ion-exchange Chromatography, IEX

By changing the salt ion strength, pH and ion exchange fillers in the protein mixture solution, different proteins have different adsorption capacities to different ion exchange resins, and proteins are separated due to different adsorption capacities or no adsorption.

Elution can be performed either by keeping the eluent composition constant or by changing the salinity or pH of the eluent. Gradient elution is generally effective with high resolution, especially for ion exchangers with small exchange capacity and sensitive to salt concentration. By controlling the eluent volume (as compared to the column volume), salt concentration, and pH, the sample components can be eluted separately from the ion exchange column.

5 Hydrophobic chromatography (HIC) for protein purification

Most hydrophobic amino acid residues are found inside proteins, but some are found on the surface. The number and spatial distribution of hydrophobic amino acid residues on the surface of a protein determine whether the protein has the ability to bind with hydrophobic column filler and use it for separation. It is a universal tool for separating and purifying proteins because of its low cost and biological activity. Protein retention in the column, in the brine solution in low salt or proteins in aqueous solution, was cleared from the column, so especially suitable for thick after precipitation separation of the mother liquor of ammonium sulfate solution, and the precipitation with salt dissolved directly into a solution containing the target product after the sample into the column, of course also apply 7 mol/L guanidine hydrochloride or 8 mol/L urea protein extract for the treatment of e. coli sample directly into the column, in the separation and the renaturation.

6. Affinity chromatography (AC) for protein purification

Combined with high efficiency, fast separation speed. Ligands can be substrates of enzymes, inhibitors, cofactors, and specific antibodies. Elution can be performed by changing the ionic strength and pH of the buffer after adsorption, or by using the same ligand solution with higher concentration or ligand solution with stronger affinity. According to the different ligands, it can be divided into:

6.1 Metal chelating medium

Transition metal ions Cu2 + and Zn2 + and Ni2 + in the form of the imine complex bonding to the stationary phase, as a result of these metal ions and tryptophan, histidine and formed the coordination bond between cysteine, thus formed the imine metal chelate - protein, make containing these amino acids of protein was the metal chelate affinity chromatography stationary phase adsorption. The stability of chelates is controlled by the dissociation constants of individual histidine and cysteine, and thus by the pH and temperature of the mobile phase.

6.2. Small ligand affinity media

Ligands are arginine, benzamide, calcium modulator, gelatin, heparin, lysine, and so on.

6.3 Antibody affinity media

The ligand has recombinant protein A and recombinant protein G, but protein A is more specific than protein G, and protein G can bind to more different sources of IgG.

6.4 Pigment affinity medium

The effect of dye chromatography depends not only on the affinity of dye ligands and enzymes, but also on the type of elution buffer, ion strength, pH value and the purity of the sample to be separated. Ligands are Cibacron Blue and Procion Red. Under certain conditions, the immobilized dyes can act as cation exchange agents. In order to avoid the occurrence of this phenomenon, it is best to operate when the ionic strength is less than 0.1 and the pH is greater than 7.

6.5. Foreign lectin affinity media

Ligands include cantaloglobulin, lentil exogenous lectin and malt exogenous lectin. The solid-phase exogenous lectin can react reverentially with several sugar residues, which is suitable for purification of polysaccharides and glycoproteins.

7. Stability

7.1 Thermal stability

Most proteins misfold or precipitate when heated to 95 ° c. this property makes it easy to separate from most other cellular proteins a protein that remains soluble when heated this way.

7.2 Proteolytic stability

The supernatant is treated with protease to digest the miscellaneous proteins, leaving resistant proteins resistant to protease hydrolysis.

GALAK ChromatographyAuthor

Tian Jing
Manager & Engineer in GALAK Chromatography. Master of Chemical Engineering.
During my college study, I found liquid chromatography to be a profound subject. I know the painful struggle a novice needs to go through to get started. I share this article to help you solve your problems quickly.

More about me >>

Does this article still not solve your problem?

Contact us now! We are 7/24 available.

As manufacture for liquid chromatography products, we provide

Scroll to Top