Question

Assume that you have no overlap between your DNA sequence and restriction sites and that you have selected the correct open reading frame. By inserting your GFP gene between BamH1 and Xho1 you will have two tags (the T7 and His) on the protein when it is expressed. What could you add to the GFP gene to only have the T7 tag?

Answer 1

An enterokinase site can be added to the C terminal of the GFP gene. So the net construct should be a T7-GFP-enterokinase-cut site sequence-Hig tag sequence. Now, this construct should have the flanking sequences of BamHI and XhoI in its N and C terminal respectively to clone into the vector.

To clone this construct vector should be cut with BamHI and XhoI and the construct should be inserted in between T7 and His tag. The protein which is expressed initially will have both of the T7 and His tags in it but during purification of the protein, enterokinase treatment is done. Enterokinase will cleave in between the GFP and His tag and will remove His-tag from it. So the protein will have only the T7 tag in its N terminal only.