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Advantages of His tag protein expression and purification

Update time : 2020-11-06

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Advantages of His tag protein expression and purification
His tag is based on the side chain of histidine residues on the protein surface. Under neutral and weakly alkaline conditions, it can interact with immobilized metal ions (such as Ni ions, Zn ions, Co ions, etc.) to achieve purified protein. It is one of the common protein purification tags at present.

1. What is His-tag?

His-tag is the most commonly used tag for recombinant protein expression and purification. Whether the expressed protein is soluble or inclusion body, it 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. Among them, the 6-histidine tag (His-Tag) is the most widely used.


The His-Tag purification tag combined with metal chelation affinity chromatography provides a powerful tool for the separation, expression and purification of recombinant proteins. 

Since metal chelation affinity chromatography has the characteristics of simple ligand, large adsorption capacity, mild separation conditions, and strong versatility, the sample loading conditions can be selected in a wide range, under the conditions of high salt, a certain concentration of denaturant and detergent Under the hood, proteins with 6His purification tags can be specifically combined with affinity fillers, gradually becoming one of the most effective techniques for separating and purifying proteins and other bioengineering products.


2. His-tag is the first choice for protein purification


(1) His-Tag is very small and has no effect on the protein structure after the fusion protein is crystallized;

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

(3) Using IMAC (immobilized metal ion affinity chromatography) to purify His-Tag fusion protein is easier;

(4) His-Tag has almost no effect on the characteristics of the target protein itself, and will not form dimers;

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

(6) Construct a dual affinity tag with other affinity tags, and can be applied to a variety of expression systems.

3. Common problems of His tag protein purification


(1) His tag protein does not bind to the medium

  • Possible cause: inappropriate ultrasound power (too large, the protein is carbonized, too small, the protein is not completely released). Solution: Change the ultrasonic power or other methods to break the cells.
  • The sample or buffer is inappropriate. Solution: Ensure that the concentration of chelating agent, reducing agent, and imidazole in the buffer is not very high.
  • The His tag is not fully exposed. Solution: Add denaturant (4-8M urea, 4-6M guanidine hydrochloride) to the buffer, and then purify with IMAC medium.
  •  The His tag is lost. Solution: If necessary, increase the number of His and ensure correct expression, while reducing the loading speed to ensure sufficient incubation time.

(2) His tag protein is difficult to be eluted on the medium

  • Possible cause: The elution conditions are too mild. Solution: increase the concentration of eluted imidazole or lower the pH.
  • Possible cause: The protein is deposited on the medium. Solution: reduce the sample load and optimize the chromatography conditions.
  • Possible cause: non-specific binding. Solution: add 2% Triton X-100 and NaCl to the buffer.

(3) The elution peak has many impurities

Possible reasons: non-specific binding, incomplete cleaning, degradation, etc. Solution: In the purification process, add protein inhibitors to prevent degradation, wash thoroughly after loading, and add a certain amount of NaCl and imidazole to reduce non-specific binding.

(4) After several times of use, the media binding efficiency and column efficiency are reduced

Possible cause: a large amount of impurities are deposited on the medium. Solution: thorough cleaning, IDA/IMAC de-nickel regeneration treatment.



Only by choosing a suitable protein expression and purification method can we obtain high-purity recombinant protein. A large number of purified recombinant proteins can be used in a series of biomedical fields such as drug screening, structural biology research, cell biology research, and proteomics. 

General Biosystems has successfully constructed a complete E. coli prokaryotic and yeast cell eukaryotic protein expression system platform, and can provide a complete set of services from protein expression plasmid construction, protein expression conditions exploration and protein expression purification according to customer needs.