Question

His-tag purification uses

• A. \( \text{Metal affinity} \)
• B. \( \text{Gel filtration} \)
• C. \( \text{Dialysis} \)
• D. \( \text{Ion exchange} \)

Hint:

Remember: His-tag = Histidine tag = Nickel/Cobalt affinity = Immobilized Metal Affinity Chromatography (IMAC). Imidazole is commonly used to elute the purified protein from the column.

Answer 1

The Correct Option is A

Concept: His-tag purification is a widely used protein purification technique in recombinant DNA technology. A short sequence of histidine amino acids (usually six histidines), called a His-tag, is attached to the target protein. This tag has a strong affinity for certain metal ions.
• His-tag generally consists of six histidine residues (\(6\times\) His).
• Histidine contains an imidazole ring that binds strongly to metal ions.
• Nickel (\(Ni^{2+}\)) and Cobalt (\(Co^{2+}\)) are the most commonly used metal ions.
• The purification technique is known as Immobilized Metal Affinity Chromatography (IMAC).

Step 1: Understand the principle of His-tag purification.
The imidazole group present in histidine has a strong affinity for immobilized metal ions such as Nickel (\(Ni^{2+}\)) or Cobalt (\(Co^{2+}\)) attached to the chromatography resin. \[ \boxed{\text{His-tag} \longrightarrow \text{Binds to } Ni^{2+}\text{ or }Co^{2+}} \]

Step 2: Understand the purification process.
When a protein mixture is passed through the metal affinity column, only the His-tagged protein binds to the metal ions. Other proteins lacking the His-tag pass through the column during washing. Finally, the bound protein is eluted using imidazole, which competes with the His-tag for binding to the metal ions.

Step 3: Choose the correct option.
Since the purification depends on the interaction between histidine residues and immobilized metal ions, the technique is based on metal affinity chromatography. \[ \boxed{Option (A) Metal affinity is the correct answer. \]