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What are the Methods of Protein Separation and Purification?

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The separation and purification of protein is an important part of biochemistry. It is a method of separating and purifying the desired target protein from the mixture using bioengineering technology. There are many protein separation companies. Medicilon is a preclinical drug development company (CRO) that can provide protein separation and purification technology services. There are many methods for protein separation and purification, mainly as follows.

Protein extraction method

(1) Aqueous solution extraction method

The aqueous solution of dilute salt and buffer system has good stability and high solubility for protein. It is the most commonly used solvent for protein extraction. The usual amount is 1-5 times the volume of the raw material. The extraction requires uniform stirring to facilitate the dissolution of the protein. The extraction temperature depends on the nature of the active ingredients. Generally, low temperature (below 5 degrees) operation is used when extracting proteins and enzymes. In order to avoid degradation during protein extraction, proteolytic enzyme inhibitors (such as diisopropyl fluorophosphate, iodoacetic acid, etc.) can be added.

(2) Organic solvent extraction method

Some proteins and enzymes that are more firmly bound to lipids or have more non-polar side chains in the molecule are insoluble in water, dilute salt solutions, dilute acids or dilute bases. Organic solvents such as ethanol, acetone and butanol can be used. It has certain hydrophilicity and strong lipophilicity. It is an ideal lipoprotein extraction solution, but it must be operated at low temperature.

Methods of protein separation and purification

Different separation methods according to protein solubility

(1) Salting out of protein

Neutral salt has a significant effect on the solubility of protein. Generally, with the increase of salt concentration at low salt concentration, the solubility of protein increases, which is called salt dissolution; when the salt concentration continues to increase, the solubility of protein decreases to varying degrees and successively This phenomenon is called salting out. A large amount of salt is added to the protein solution. The high concentration of salt ions (such as SO4 and NH4 of ammonium sulfate) have strong hydration power, which can seize the hydration layer of protein molecules and make The “loss of water”, so the protein colloids coagulate and precipitate out. If the pH of the solution is at the isoelectric point of the protein during salting out, the effect will be better. Because the particle size and hydrophilicity of various protein molecules are different, the salt concentration required for salting out is also different. Therefore, adjusting the neutral salt concentration in the mixed protein solution can make various proteins precipitate in stages.

(2) Isoelectric point precipitation method

When the protein is in an electrostatic state, the electrostatic repulsion between particles is the smallest, so the solubility is also the smallest. The isoelectric points of various proteins are different. You can adjust the pH of the solution to reach the isoelectric point of a certain protein to make it precipitate, but this method is very It is rarely used alone, and can be used in combination with the salting-out method.

(3) Low temperature organic solvent precipitation method

Use water-miscible organic solvents such as methanol, ethanol or acetone to reduce the solubility of most proteins and precipitate out. This method has higher resolution than salting out, but proteins are more susceptible to denaturation and should be carried out at low temperatures.

Separation method according to the difference of protein molecule size

(1) Dialysis and ultrafiltration

Dialysis uses a semi-permeable membrane to separate proteins of different molecular sizes. The ultrafiltration method uses high pressure or centrifugal force to force water and other small solute molecules to pass through a semi-permeable membrane, while the protein remains on the membrane. You can choose different pore sizes to intercept proteins of different molecular weights.

(2) Gel filtration method

Also called size exclusion chromatography or molecular sieve chromatography, this is one of the most effective methods for separating protein mixtures based on molecular size. The most commonly used packing materials in the column are glucose gel (Sephadex ged) and agarose gel (agarose gel).

Separation according to the charged properties of the protein

Proteins have different charged properties and different amounts of charge in different pH environments, and they can be separated.

(1) Electrophoresis method  

Under the same pH condition, various proteins can be separated due to their different molecular weights and different charges in the electric field. It is worth paying attention to isoelectric focusing electrophoresis, which uses an amphoteric electrolyte as a carrier. During electrophoresis, the amphoteric electrolyte forms a pH gradient that gradually increases from the positive electrode to the negative electrode. When a certain charged protein swims in it, it will reach each other. The pH position of the electrical point stops, and this method can be used to analyze and prepare various proteins.

(2) ion exchange chromatography

Ion exchangers include cation exchangers (such as carboxymethyl cellulose, CM-cellulose) and anion exchangers. When the separated protein solution flows through the ion exchange chromatography column, it has a charge opposite to that of the ion exchanger. The protein is adsorbed on the ion exchanger, and then the adsorbed protein is eluted by changing the pH or ionic strength.

Separation method based on ligand specificity-affinity chromatography

Affinity chromatography is an extremely effective method for separating proteins. It often requires only one step to separate a certain protein to be purified from a very complex protein mixture with high purity. This method is based on the ability of certain proteins to bind specifically but not covalently to another molecule called a ligand. The basic principle: protein exists in the form of a complex mixture in tissues or cells, and each type of cell contains thousands of different proteins.

Most proteins are soluble in water, dilute salt, dilute acid or alkali solution, and a few proteins bound to lipids are soluble in organic solvents such as ethanol, acetone, butanol, etc. Therefore, different solvents can be used for extraction, separation and purification. Protein and enzymes. There is no single or a set of ready-made methods that can extract any kind of protein from a complex mixture of proteins, so several methods are often used in combination.

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