His-tagged Proteins

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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
	His-tag is not present	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
	His-tag is inaccessible	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
	His-tag is partially hidden	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 precipitation	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
	Protein precipitation	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
	His-tagged proteins are associated with contaminants	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.