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Suzhou protein purification system: protein purification methods and their advantages and disadvantages (3)

Suzhou protein purification system: protein purification methods and their advantages and disadvantages (3)

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  • Time of issue:2020-03-05 15:58
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(Summary description)Suzhou protein purification system: protein purification methods and their advantages and disadvantages (3)

Suzhou protein purification system: protein purification methods and their advantages and disadvantages (3)

(Summary description)Suzhou protein purification system: protein purification methods and their advantages and disadvantages (3)

  • Categories:Industry news
  • Author:
  • Origin:
  • Time of issue:2020-03-05 15:58
  • Views:
Information

I will talk about the affinity chromatography of protein purification methods with you today. Below is the editor of SCG protein purification system.

Affinity chromatography is based on the specific binding of the target protein to the solid phase ligand, and other contaminants will flow through the column. The problem with this method is that the monoclonal antibody is very expensive and needs to be purified first; the binding capacity of the monoclonal antibody to the target protein is too strong. It must be eluted under harsh conditions, which will inactivate the target protein and destroy the monoclonal antibody; mixture Other proteins in the protein, such as proteases, may also destroy antibodies or bind them non-specifically; some monoclonal antibodies will also dissociate from the resin during the purification process and mix into the product, which also needs to be removed from the final product. Affinity columns are usually used late in the purification process, when the specimen volume has shrunk and most of the impurities have been removed.

Glutathione S-transferase (Glutathione S-transferase, GST) is one of the commonly used affinity chromatography purification tags. Recombinant proteins with this tag can be purified by cross-linked glutathione chromatography media, but the original The method has the following disadvantages: first, the GST on the protein must be properly folded to form a spatial structure that binds to glutathione to be purified by this method; second, the GST tag has up to 220 amino acids, such a large tag may affect The solubility of the expressed protein leads to the formation of inclusion bodies, which will destroy the natural structure of the protein and make it difficult to analyze the structure. Sometimes, even after purification, the GST tag may not be removed by digestion.

Another applicable affinity purification tag is the 6-histidine tag. The imidazole side chain of histidine can affinity bind metal ions such as nickel, zinc, and cobalt with histidine under neutral and weak alkaline conditions. The target protein of the tag is bound to a nickel column and is eluted with imidazole at low pH. Compared with GST, histidine tags have many advantages. First, because there are only 6 amino acids, the molecular weight is very small, and generally need to be removed by enzyme digestion: second, the protein can be purified under denaturing conditions, and it can still be used in high concentrations of urea and guanidine Maintain the binding force; the other 6 histidine tags are not immunogenic, and the recombinant protein can be directly used to inject animals without affecting immunological analysis.

Although there are so many advantages, this tag still has shortcomings, such as the target protein is easy to form inclusion bodies, difficult to dissolve, poor stability and misfolding. During the purification of the nickel column, the metal nickel ions are easy to fall off and leak into the protein solution. Not only will the amino acid side chain of the target protein be destroyed by oxidation, but the column will also specifically adsorb the protein, affecting the purification effect. If the protein of interest can be specifically bound to a certain carbohydrate, or a special cofactor is needed, the carbohydrate or cofactor can be solid-phased to form an affinity column. After binding, the target protein can be used with a high concentration of carbohydrate or cofactor Factor elution.

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Time of issue:2020-02-24 00:00:00

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