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His-tagged Proteins

Recombinant proteins have shown great differences in solubility, stability, and functionality during their expression and purification, which makes it difficult to perform large-scale protein production. Addition of fusion tags is the best way to effectively improve the expression level, solubility, and stability of recombinant proteins, especially for proteins that is difficult to express.

His-tag
The most frequently used fusion tag to collect large amounts of high-quality protein is the His-tag. It usually composes of 6-14 histidines, to which the N- or C-terminal of the target protein is typically fused. The His-tag contains imidazole side chains for irreversibly engagement of coordinative bonds to Ni2+, Co2+, or Zn2+. Ni2+ is preferred because of the greatest affinity and selectivity for His-tags. His-tagged proteins prefer to form a considerably stronger bond to the Ni-NTA resin than any endogenous protein that contains histidine. How many histidines bind to the matrix directly affect the relative binding strength.

Advantages of His-tag:
1. Easy control of the number of the histidine in a His-tag. The longer His-tag is, the better binding and separation it delivers;
2. His-tags are much smaller than other fusion tags;
3. Purification can be performed under native and denaturing conditions;
4. Increase the solubility of target proteins;
5. Rarely interfere with function of target proteins.

His-tagged Protein Purification Troubleshooting

Problem Reason Solution

His-tagged proteins do not bind to Ni-NTA

His-tag is not present

Check the reading frame to ensure it is correct
Check for potential internal translation or premature termination sites
His-tag degradation Make sure that the His-tag is not in association with a portion of the processed protein

His-tag is inaccessible

Perform His-tagged protein purification under denaturing conditions
Move His-tag to the other terminus of the target protein
Incorrect binding conditions  Check all the buffers and solutions (pH and compositions) 
His-tagged proteins present in the Wash Buffer His-tag is partially hidden Perform His-tagged protein purification under denaturing conditions
Reduce washing stringency
Incorrect binding conditions  Check all the buffers and solutions (pH and compositions) 
Wash stringency is too high Increase the pH slightly or decrease the concentration of imidazole

His-tagged proteins precipitation

Low temperature Perform His-tagged protein purification at room temperature
His-tagged proteins aggregation Add solubilization reagents  (0.1 % Triton X-100, or up to 20 mM βME, or  0.1 % Tween-20)
His-tagged proteins  do not elute Mild elution conditions Optimize elution conditions of pH or imidazole concentration

Protein precipitation

Perform binding and elution in batch format
Perform elution under denaturing conditions
Poor purity of His-tagged proteins His-tagged proteins are associated with contaminants  Add up to 20 mM βME to reduce disulfide bonds
Increase salt/detergent concentration
The amount of Ni-NTA resin is too large Reduce the amount of Ni-NTA resin
Binding/washing condition is insufficient stringent  Add 10-20 mM imidazole in the binding/wash buffer
Resin color turns white Chelating compounds present in buffers Remove chelating compounds
Resin color turns brown Reducing agents present in buffers Remove reducing agents

Creative Biostructure is happy to accelerate your His-tagged protein research project from plasmid construction to high-quality recombinant protein production. Please see the table below for more information and feel free to Contact Us.