Poly-histidine (His)-tagged proteins are commonly used in recombinant protein expression and purification. The His-tag offers advantages such as small size, high affinity for binding to IMAC (Immobilized Metal Affinity Chromatography) resins, and one-step purification. However, there are instances when a His-tagged protein fails to bind to the affinity column, leading to frustration. In this article, we discuss troubleshooting steps to address this issue.

Binding of His-tagged proteins to surfaces functionalized with NTA

Binding of His-tagged proteins to surfaces functionalized with NTA

 

Common Challenges and Solutions

1. Solubilization Buffer Optimization

Problem: Ni-NTA resins are sensitive to reducing agents (e.g., DTT) and chelating agents (e.g., EDTA or EGTA). These substances can affect the binding capacity of IMAC resins.

Solution:

Ensure that solubilization buffers do not contain reducing or chelating agents.

Maintain an imidazole concentration of 15-25 mM in the binding buffer to prevent non-specific protein binding.

Adjust the pH of the buffer close to the proteinā€™s isoelectric point.

2. Fresh and Functional Affinity Resin

Problem: Using old or compromised Ni-NTA resin can lead to poor binding.

Solution:

Always use fresh Ni-NTA resin, especially when purifying a novel protein for the first time.

Perform batch binding with a control His-tagged protein (previously purified using IMAC) alongside your protein of interest. If the control protein binds but your protein does not, investigate further.

 

3. Hidden His-Tag

Problem: Some proteins bury the His-tag within their folded native structure, rendering it inaccessible for metal coordination.

Solution:

Test binding in the presence of denaturing agents (e.g., urea or guanidinium chloride). If the His-tagged fusion protein binds under denaturing conditions, hidden tag folding may be the issue.

Options:

Purify the protein under denaturing conditions and then refold it.

Add a flexible linker (e.g., poly-serine or poly-glycine) to separate the tag from the fusion protein.

Try placing the His tag at the opposite terminus of the construct.

 

4. Non-Optimal Binding Buffer

Problem: Buffer composition affects His-tag binding.

Solution:

Pay attention to imidazole concentration and pH in the binding buffer.

Optimize buffer components to enhance binding efficiency.

Alternatives tags:

Glutathione-S-Transferase (GST) Tag: for purification and pull-down assays

Biotin Tag: for immobilization or detection

Maltose-Binding Protein (MBP) Tag: enhances solubility – for affinity purification and protein-protein interaction studies

Strep-TagĀ® II: high-affinity binding to streptavidin – for purification and detection applications

FLAGā„¢ (DYKDDDDK) Tag: for detection, immunoprecipitation, and Western blotting

SUMO Tag: removable ā€“ to produce native N-term protein

Myc Tag: removable ā€“ for purification, detection and crystallization

Fc Tag: IgG constant domain ā€“ for ELISA detection, affinity purification and increase expression

 

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