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) 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. How you will elute the HIS-tagged protein from the column? a. C. 76

Answer 1

A)A hexa-histidine tag is an amino acid sequence conformed by 6 his residues in a row. This amino acid is responsible several process such as degradation of GM2 gangliosides in the brain. His tag can be introduced in the N-terminal segment of a protein by molecular biology techniques such as a expression vectors where a plasmid is used to introduce the specific gene of the protein and the hexa-histidine tag to produce the recombinant protein.

B)The His tag purification is made by immobilized metal affinity chromatography. First, transition metal ions (Ni2+ , Cu2+ or Zn2+ )are immobilized on a resin matrix. Second, the his-tag has a high affinity for these ions so they bind strongly to the column.

C)To elute the HIS-tagged, you should add a low concentration of Imidazole (which competes with the his-tag for binding to the metal resin) and wash buffer to the column to interfere with the weak binding of other proteins. Once this process is done, you should had a higher concentration of imidazole to obtain the HIS-tag.