Question

12. In some instances, using a constitutively active promoter to drive the expression of your gene is not preferable. Why?

13. In some instances, it is preferable to tag a protein with a marker such as FLAG instead of GFP. Why?

14. Other than GFP and FLAG, list 2 other markers that are commonly used to tag proteins.

Answer 1

12) A consitiutive active promoter is acitve all time , therefore the gene downstream of it will be expressed all time. In cases where gene encodes a protein which can be toxic to the host cells at high levels, there is a need to use promoter which are regulatable to prevent leaky expression. Also promoter should be tightly regulated to allow the cells to reach high cell density. If promoter is constitutive cells energy will be diverted to protein expression than cell growth.

13) GPF is a reporter gene to check if our gene of interest in being expressed in the host cell or not. It is an autofluorescent protein which emits green light.

FLAG marker on the other hand is useful for protein purification purpose or studying protein-protein interactions. It is a short hydrophilic protein against which anti-FLAG monoclonal antibodies are available. FLAG can be expressed as a fusion partner to our protein of interest. This helps in purification of protein of interest or studying interaction of the protein with other using pull down assay.

14) GST and His₆ are other two commonly used markers to tag proteins. GST is glutathione-S-transferase which binds to its substrate glutathione. Glutathione can be immobilized on solid support to aid in purification of GST tagged proteins. GST tag is generally attached to the N-terminus of a protein

His₆ consist of 6 histidine residues which can be attached to either N or C terminus of a protein. It binds to Ni²⁺ metal ion bound to solid support column.