7. You are interested in studying a novel protein (Protein X) discovered in your

Question

7. You are interested in studying a novel protein (Protein X) discovered in your research lab. You have engineered a version of the protein that contains a HiS-tag (hexa-histidine). Devise a reliable procedure for its purification from a cell lysate. Answer the following questions. (15 points) a. What is a hexa-histidine tag? How do you introduce a HiS-tag to your protein of choice? b. Indicate the type of column and how it will work to purify HIS- tagged Protein X c. How you will elute the HiS-tagged protein from the column?

Explanation / Answer

7.

a. Hexa-histidine tag is a hexapeptide of histidine attached with the protein of interest at one of its terminals. His-tag can be introduced in the protein by synthesising recombinant gene that contains six histidine coding codons (before or at the end) along with the gene for protein of interest.

b. Ni-NTA (Nickel-nitrilotriacetic acid) affinity gel column is used to purify His-tagged Protein X. The gel has affinity for binding with histidine, so it will bind with His-tagged protein in the sample strongly while other non-specifically binded proteins can be washed.

c. His-tagged protein can be eluted by using high concentration of imidazole. As histidine has imidazole ring in its structure, elution with high concentration of imidazole results in disruption of Interaction between affinity gel and His-tag.

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