There is no reason why it should not work except for exposure of the His Tag. You can add some flexible linkers between the two proteins and the His tag to increase the chance of exposure for the His tag. Commonly used flexible linkers are generally Gly Ser rich, for example (GGS)4, ...

David- why relocate the tag from the N-terminus to an internal site? You want the tag to be as highly accessible as possible, which is more likely to be true at either the N- or C-termini. While in principle, the internal his-tag might work if it falls in an unstructured domain, you run the risk of protein folding or aggregation impeding the function of the tag (unless, of course, you opt for a denaturating purification protocol).

I know the internal tag location will be outside the native structure (should be fully accessible). Also originally I was going to purify in 8M urea, but I might also test native conditions. The aggregation prone protein is very aggregation prone and I might need the bacterial chaperone system to help fold the soluble protein tag.

I suppose you can't lose anything by trying, so it might make sense to test out both designs. N-terminal his-tags perform extremely well in my experience, and are relatively easier to clone; however, I suppose this depends on your cloning strategy for introducing the cDNA for your Soluble Protein into your expression vector (that is, whether the soluble protein cDNA or your vector already contain a suitable his-tag sequence).

M-6histag-SolubleProtein-Thrombin-AggregationProne protein is a good way than the later. I don't have first hand experience with such a construct but I would go with first method. Soluble protein-Thr-AggProne-His tag also good idea. I used MBP-3C-myprotein-His8 and purified on Ni beads gave me purest protein when compared to MBP-3C-myprotein on Amylose resin. This is not universal. But you should try to know what is good. Also, when I did this I didn't know how to cleave C-terminal his tag but now I guess using carboxy peptidase (need to add proline after the native amino acids so that peptidase can stop at Proline, need to check exact details) we can remove c-terminal his tag (crystallographers don't like extra residues).

I am going to try the N-terminal tag first. I know the soluble protein expresses well with the His tag on the C-terminal side (50-90mg/L) so hopefully I will get the same with the tag on the N-terminal side. When something really works, I do find it hard to change.

I asked this question as from a Google search, I couldn't find anyone whom has placed the His tag between the proteins. In my design I know this is very flexible region.

Thank you for the reassurance that a N-terminal tag should work.

How many should the number of base pairs be included after the addition of his-tag for his-tag primer design?Is it ok if the number of base pairs after his-tag sequences is around 30 bp.?

Hi David. Our lab has successfully used the pET32b vector system for protein purification, and I believe that's essentially what you are describing. An internal Hisx6 tag works perfectly fine, It might not hurt to model your vector off of this system. I attached the vector map for you.

I have had good expression with the Soluble-His-Protease site-Target protein construct. I've been using the pET48-50b vector series, which have Trx/GST/NusA-spacer-His6-HRV3C-MCS sequences. I also dropped maltose binding protein in this setup as well, so I have a series of vectors with the same MCS/antibiotic/promoter and drop my insert into each (or at least 2-3) and see which gives the best soluble expression. The Novagen/EMD vectors have a polylinker between the N-terminal fusion and the His-tag which helps with both His-tag and protease site accessibility. As SD mentioned, getting rid of the C-terminal tag can be tricky, and having N- and C-terminal tags means you have to lop off both if you want native target sequence.

Also, I've never had consistent cleavage with thrombin (or Ek or FXa for that matter). I have become a big fan of Prescission/HRV3C which, for me at least, has worked very well with no nonspecific cleavage even after a weekend of digestion. TEV is also pretty good.

Place the 6His-tag in a loop between proteins it may help. To make a loop, flank 6His tag by glycines or prolines .

Thank you David Horita for the comments on Protease. I used thrombin in the past and it worked. But I think I might try TEV as it only will add a Gly to my target, and its digestion conditions are ideal for my application.

The His tag capacity depends on its exposure. it doesnt matter where is it located. if you protein folds in such a way that mask the histidines then you cant purify. If you have some idea about folding and confirmation in purifying conditions of your fusion protein then you can guess otherwise, just give a purification trial. If you hear about histidine rich glycoprotein HRG!, that is present in human serum. This protein has several His repeats in the middle of the protein sequence. It can perfectly bind to Ni-NTA resin in presence of 80-100 mM imidazole and then eluted at 200 mM imidazole. it is just one step purification, you can get >90% purified HRG.

There is no reason why it should not work except for exposure of the His Tag. You can add some flexible linkers between the two proteins and the His tag to increase the chance of exposure for the His tag. Commonly used flexible linkers are generally Gly Ser rich, for example (GGS)4, ...

Internal His tags generally work. How well depends on your proteins before and after. If you encounter low binding on affinity column, make 12xHis. you cleave everything off anyway, right? In principle, 3-4 exposed His should suffice for high affinity.

I've seen people using three tags in one construct, partially for purification and partially for solubility. SUMO is a great tag for solubility enhancing and specifically cleaved by the SUMO protease.

I am trying to make a construct with the His tag placed at the C terminus right after the MBP, in this way:  Aggregation protein_enterokinase cleaving site_MBP_6X His tag

  Is this a good strategy?

This could work.  Do you know if the N or C terminus is involved in the aggregation pathway of the protein?  If so I would put the tag on the end at is aggregation prone.

Since I asked this question I was able to take a really soluble protein (TTR) add it to the N-terminus of an extremely aggregation prone peptide, separated by a TEV digestion site and get excellent yield and solubility.  Construct was M-6his-TTR-TEV-Ure2(1-100), the N-terminus is involved in aggregation and my yield was 8mg/mL.  I don't think you can purify any soluble Ure2 (1-100), as its that aggregation prone. It even aggregates in 8M Urea and in Gdn. 

A tested source of genetically modified TEV variant with improved stability and activity is here: https://www.protean.bio/en/recombinant-protein/28/tev-protease

Unless it is necessary putting tag in middle of proteins not advisable. The tag could hinder proper folding of recombinant molecule.