I suggest the second one. If you have the ATG between His-tag and your gene alternative translation initiation may happen, so you can have proteins without His-tag, too. However, I worked with constructs like the first one and there was no problem.

But if you can choose, choose the second one.

Best,

F

I suggest the second one. If you have the ATG between His-tag and your gene alternative translation initiation may happen, so you can have proteins without His-tag, too. However, I worked with constructs like the first one and there was no problem.

But if you can choose, choose the second one.

Best,

F

Dear Farkas and Thomas, I agree with you and indeed I always remove start codon of gene and always prefer to use vector-brone start codon which is present upstream to His tag. However I am little bit worried about protein structure and function. Is there any possibility of alteration/loss of function due to this removal? Moreover, I have not found any reference/book/protocl which favors the removal of ATG of coding sequence.

You can use either solution, as discussed above. I never observed significant production of untagged protein arising from the second potential start codon, it is way too far from the Shine-Dalgarno sequence. Since you use the His tag for purification, only the protein translated from the first start codon will bind to the affinity resin.