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Advantages and principles of protein purification His-tag

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1.What is His-tag?

His-tag monoclonal antibody is also called 6His-tag monoclonal antibody, or 6His monoclonal antibody. It is prepared with mice. His can be adsorbed on a nickel column and used to purify recombinant proteins, regardless of whether the expressed protein is soluble or Inclusion bodies can be purified by immobilized metal ion affinity chromatography (IMAC). Metal chelation affinity chromatography, also known as immobilized metal ion affinity chromatography (IMAC), is a new type of separation technology developed in the past 30 years. It was first proposed by Paroth et al. This method uses the principle that some amino acids on the protein surface, such as histidine, tryptophan, and cysteine, can interact with metal ions to separate the protein. These functions include coordinate bonding, electrostatic adsorption, and covalent bonding. Among them, coordinate bonding is the main one, and among them, the 6-histidine tag (His-Tag) is the most widely used. His-tag is a tag often used in protein recombination technology. Its sequence is 6 histidines HHHHHH. It is characterized by a small molecular weight, less than 0.84 KD. It basically does not change the biological structure of the protein, and does not change the protein’s structure. Solubility, and more importantly, it makes the purification of proteins extremely convenient. According to the principle that the imidazole ring on histidine can bind to divalent metal ions, His-Tag can be bound to the C-terminus or N-terminus of the target protein to form a special structure for the next step of purification and detection. People can use metal ion affinity chromatography to purify proteins with a His-tag, that is, the lysate containing the target protein is passed through a fixed divalent metal ion (usually divalent Ni ion) filler, with 6*his-tag The protein is bound to the filler, and other proteins are not bound to the filler. Finally, the target protein can be eluted with a high concentration of imidazole.

His-tag monoclonal antibody prepared with experimental mice as a host is called His-tag mouse monoclonal antibody. The preparation method is usually as follows: artificially synthesize 6*His polypeptide, that is, a polypeptide with an amino acid sequence of HHHHHH, and add special amino acids used in the coupling carrier to the end if necessary. After synthesizing the polypeptide, chemically couple the polypeptide with the carrier protein (such as KLH, BSA, OVA, etc.). After the coupling is completed, immunize the experimental mice with the coupled whole antigen, and kill after the immunization In the immunized mice, the spleen is taken under aseptic conditions, fused with myeloma cells, and positive clones are screened by ELISA or other means. The selected cells form a stable cell line after cloning. The strain is cultured in vitro or induced ascites formation in mice, and then purified from the culture medium or ascites to obtain His-tag mouse monoclonal antibody, which can be identified by Western blot or other means.


2. Advantages of His-tag becoming the first choice for protein purification:

His-Tag is gradually becoming one of the most effective technologies for separating and purifying proteins and other bioengineered products. His-tag monoclonal antibody can be used to identify the expression of His-tag protein (such as the relative expression and molecular weight of the target protein), location in the cell, and other characteristics.

His-tag is the first choice for protein purification. Its advantages are:

(1) The N-terminal His-Tag is compatible with the transcription and translation mechanism of bacteria, which is conducive to protein expression;

(2) Using IMAC (immobilized metal ion affinity chromatography) to purify His-Tag fusion protein is more convenient;

(3) His-Tag has almost no effect on the characteristics of the target protein itself, and will not change the solubility and biological functions of the target protein itself;

(4) His-Tag is very small, generally does not affect the function of the protein, and has no effect on the structure of the protein after the fusion protein is crystallized;

(5) The immunogenicity of His-Tag is relatively low, and the purified protein can be injected directly into the animal body for immunization and antibody preparation;

(6) It is constructed into a dual affinity tag with other affinity tags, and can be applied to a variety of expression systems, with mild purification conditions; His-Tag fusion protein has a wide range of applications, and can exist in non-ionic surfactants. Purification can also be carried out under denaturing conditions. The former is usually used to purify highly hydrophobic target proteins, while the latter is usually used to purify inclusion body proteins.

3.Principle of His-tag protein purification:

His-Tag can have special interactions with a variety of metal ions, such as Ca2+, Mg2+, Ni2+, Cu2+, Fe3+, etc. The principle is to use the characteristics of the protein surface to be adsorbed on the gel column to achieve separation and purification. The purpose of the protein. The histidine (His) residue has an imidazole group, which can form coordination bonds with transition metal ions such as Ni2+ and Co2+ and selectively bind to metal ions. These metal ions can use chelating ligands It is fixed on the chromatographic medium, so the protein with His-Tag can be selectively bound to the medium when it passes through the chromatographic medium equipped with metal ions, while other impurity proteins cannot bind or only weakly bind. The His-Tag protein bound to the medium can be eluted competitively by increasing the imidazole concentration in the buffer, thereby obtaining a higher purity His-Tag protein. The most widely used in affinity purification experiments is Ni2+. According to the different binding groups, Ni2+ affinity chromatography columns can be divided into two categories: one is Ni-IDA and the other is Ni-NTA. Ni2+ has six chelation valences, among which Ni-IDA chelates trivalent and Ni-NTA chelates quadrivalent. Therefore, the loading capacity of IDA is higher than that of NTA. Under the same conditions, the imidazole concentration required for the elution of Ni-IDA is also higher than that of Ni-NTA. However, its weak binding force makes it easy for metal ions to be leached during the elution stage, and tightly binds to the target protein, which leads to problems such as low yield of isolated protein, impure product and metal ion contamination. NTA has uniform particle size and smaller particle size, and chelated nickel is more stable, can tolerate higher reducing agents, makes the filler more stable, and nickel ions are not easy to fall off, so Ni-NTA affinity chromatography column is selected for protein purification.

(His-tag: Purified boutique His histidine tag fusion protein)

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