Take a look on your vector map, His-taq is a part of your vector , upstream or downstream or both of your site of restriction enzyme. 

If you generate your coding sequence by de novo gene synthesis, the possibility of an N-terminal His tag would be feasible whilst also eliminating the thrombin-cleavable S-tag present in the existing pET29 sequence.  There is an XbaI site in pET29 - see the attached map.  You could use the XbaI site upstream of the MCS providing that all of the functional residues (including RBS etc) necessary for transcription from the T7 promoter are included in the synthetic sequence upstream of a new N-terminal Methionine, His-tag in frame with your sequence are present.  Alternatively, clone into the NdeI site at the 5' end with a 3' XhoI site, with or without a stop codon depending on where you want the vector integral His tag to be used or not.

None of this is particularly difficult - please feel free to discuss further here.

Thanks a lot dear Robin and Mangal. I already removed the XbaI site of the PET29C. I do have two insertions as my GoI will be fused in another gene sequence in the vector which includes XbaI site.  However, the first restriction enzymes that I am using are NdeI and KpnI. These sites were added in the ends of my primers so my insert has them on the sides.

Another point is that my protein doesn't have the stop codon either.

so, with this situation, is it possible to use his tag of the vector for protein purification?

Best of wishes,

As I see that in pET29C+ the both  Kpni and Ndei sites are outsite the MCS, my insert wont get tagged?

thanks.

Hi Mahbobeh,

apologies for the long delay in responding. In principle, assuming your insert was cloned in frame into the NdeI / KpnI sites, without the stop codon it will read through the MCS. As you are using pET29C instead of the A version, the His- tag will not be in frame with the NdeI / KpnI insert unless you have included extra bases to push the MCS into frame with your insert. If you undertook the same cloning (NdeI / KpnI) with pET29a, the C-terminal His-tag would be in frame with your insert although with several AA of padding from the MCS being present.

As there has been a delay in response, what decisions did you make with the cloning process? Have you encountered any other issues since that we can assist with? Best wishes,