Question 17 of 21intermediate🔧 ApplyLong Answer5 marks

If you are given a recombinant protein with 6-His-tags, how are you going to use it? Explain the methodology and application in detail.

Correct Answer

A recombinant protein with 6-His-tags can be used primarily for protein purification. The 6-His-tag is a peptide consisting of six consecutive His amino acids that has the unique property of binding to metal ions like Nickel, Zinc and Cobalt with great affinity at mildly alkaline pH.

The methodology involves affinity chromatography. The DNA encoding for the 6-His-tag is fused with the gene sequence of the recombinant protein of interest. During the purification process, the 6-His-tagged recombinant protein gets immobilised by the presence of metal ions in the agarose gel.

After immobilisation, all other proteins are removed by washing the gel. This ensures that only the desired tagged protein remains bound to the column. The 6-His-tagged recombinant protein can then be eluted by using an elution buffer, resulting in a purified protein.

The main application of 6-His-tagged recombinant protein is for the ease of purification. This technique allows for efficient and easy purification of recombinant proteins, which is essential for various downstream applications in biotechnology. The purified proteins can then be used for developing novel reagents, diagnostics, and therapeutics.

Exercise: EXERCISES | Q: 17 | (Page 29)
For More Understanding

Explanation

The question asks about the use of a recombinant protein with 6-His-tags. According to the textbook context, the 6-His-tag is a peptide with six consecutive His amino acids that binds to metal ions (Nickel, Zinc, Cobalt) with great affinity at mildly alkaline pH. This property is exploited for protein purification through affinity chromatography. The methodology involves fusing DNA encoding for 6-His-tag with the gene sequence of the recombinant protein, immobilising it on metal ion-containing agarose gel, washing away other proteins, and eluting with elution buffer. The application is primarily for ease of purification of recombinant proteins.

Solution Steps

  1. Step 1: Identify that 6-His-tag binds to metal ions (Ni, Zn, Co) at mildly alkaline pH

  2. Step 2: Explain the methodology - DNA encoding 6-His-tag is fused with gene of interest

  3. Step 3: Describe affinity chromatography process - immobilisation on agarose gel with metal ions

  4. Step 4: Explain washing to remove other proteins and elution using elution buffer

  5. Step 5: State the application - ease of purification for downstream biotechnology applications

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