His tag protein purification | Application of his tag purification | Affinity chromatography

Summary

This video script delves into His-tag protein purification, a method utilizing single-step affinity chromatography with a nickel-nitrilotriacetic acid (Ni-NTA) matrix. The technique leverages the specific interaction between the matrix and a hexahistidine tag attached to the protein of interest, enabling efficient separation from a protein mixture. The process involves equilibration, binding, washing, and elution steps, ensuring the purification of the native protein. Advantages include compatibility with cell culture media, ease of use, sensitivity, and cost-effectiveness, though some optimization may be necessary to minimize non-specific binding.

Takeaways

• 🧪 His-tag protein purification utilizes a single-step affinity chromatography method with immobilized metal ion chromatography (IMAC).
• 🔗 The purification relies on the specific interaction between a nickel-nitrilotriacetic acid (Ni-NTA) matrix and a hexahistidine tag attached to the protein of interest.
• 🔬 The hexahistidine tag is incorporated at the DNA level, either at the N-terminal or C-terminal of the open reading frame, and then expressed in bacterial or mammalian cells.
• 📦 The workflow of His-tag purification includes equilibration, binding, washing, and elution steps to separate the tagged protein from a mixture.
• 🌡️ Equilibration buffer must be compatible with the protein of interest, considering factors such as ionic strength and pH to maintain protein stability.
• 🔒 During the binding step, the tagged protein binds to the Ni-NTA matrix through non-covalent interactions like coordinate bonds, while other proteins in the lysate do not bind.
• 🚿 The washing step removes non-specifically bound proteins, ensuring that only the specifically bound tagged protein remains on the column.
• 🌟 The elution step involves using a buffer with altered pH and ionic strength to release the bound protein, which can then be collected.
• 🛑 Post-purification, techniques like SDS-PAGE and Western blot can be used to verify the success of the purification process.
• 💰 His-tag purification is cost-effective compared to other chromatographic techniques such as gel filtration or HPLC.
• ⚙️ Optimization may be required to minimize non-specific binding, which involves adjusting the ionic strength of the buffers used in the process.
• 📚 Additional resources and flashcards on this topic can be found on the instructor's social media platforms, with links provided in the video description.

Q & A

What is His-tag protein purification?

His-tag protein purification is a method that allows for the single-step purification of proteins using immobilized metal ion affinity chromatography with a nickel-nitrilotriacetic acid (Ni-NTA) matrix. The protein is tagged with hexahistidine, which interacts specifically with the matrix.

How is the His-tag attached to the protein?

The His-tag is attached to the protein by cloning the open reading frame with a hexa-histidine tag at the N-terminal or C-terminal into an expression vector, which is then introduced into bacterial or mammalian cells to produce the tagged protein.

What is the principle behind the separation in His-tag protein purification?

The principle behind the separation is the specific interaction between the hexahistidine tag on the protein and the nickel ions on the Ni-NTA matrix, which involves non-covalent interactions like coordinate bonds.

What are the steps involved in the His-tag protein purification process?

The steps are equilibration, binding, washing, and elution. Equilibration involves running a buffer through the column, binding is where the tagged protein attaches to the matrix, washing removes non-specific interactions, and elution collects the purified protein.

Why is the equilibration buffer important in the purification process?

The equilibration buffer is important because it ensures that the column material is soaked and equilibrated at a specific pH that is compatible with the protein of interest, preventing denaturation.

What factors should be considered when choosing the equilibration buffer?

Factors to consider include ionic strength and pH, as these can affect protein stability and prevent denaturation during the purification process.

How does the binding step in His-tag purification work?

In the binding step, the protein sample, usually a cell lysate, is loaded onto the column. The tagged protein binds to the Ni-NTA matrix through non-covalent interactions, specifically coordinate bonds.

What is the purpose of the washing step in the purification process?

The washing step ensures that any non-specific, untagged proteins are removed from the column, breaking down weak bonds and retaining only the specific interactions between the His-tag and the matrix.

How is the elution step different from the other steps in His-tag protein purification?

The elution step involves using a buffer with altered pH and ionic strength to loosen the binding between the matrix and the protein, allowing the purified protein to be collected.

What methods can be used to verify the success of His-tag protein purification?

SDS-PAGE and Western blot can be used to verify the purification by checking the presence and purity of the protein of interest.

What are some benefits of using His-tag protein purification compared to other techniques?

Benefits include compatibility with cell culture media and lysates, ease of use, sensitivity, specificity, and being more cost-effective compared to techniques like gel filtration chromatography or HPLC.

What are some potential disadvantages of His-tag protein purification?

Disadvantages include the need for optimization to minimize non-specific binding, which may require calibration of the ionic strength of the equilibration or binding buffer.