I'm not of the same opinion of Rohan: it depends from the tag you are going to use. If you choose a 6His-tag it should be placed at N-terminal and usually it's not necessary to cleave it out because for its poor antigenicity so it will not affect your downstream experiments. Moreover, placing it at N-terminal will decrease the probability to have protein products without tag, since if you place it at C-terminal truncated polypeptides could not be purified. A proof of this is the majority of commercial plasmids that have tags right before the polylinker. Anyway, speaking about His tag, the first choice is the N-one, 2nd choice the C-one, 3rd choice the N- and C- ones......... 4th choice, forget about it.

Despite the fact that you got two strong opinions on C- and N-, I will agree with "it depends". The best tag position is basically determined by your protein's 3D structure, as you need the tag to not interfere with it. A less likely problem but also important is that you also need the tag to be exposed during the purification, and if it ends up folded in the protein's interior you will not be able to do a purification under native conditions. While for most proteins a 3D structure is not available, surely you know (or can at least use some tools to predict) where the important domains are, and avoid them.

One last thing, if you have commercial vectors for these tags, do not assume that a "His-tag" vector means a His- tag ALONE is added on your protein - check the exact maps as the actual aminoacids added may be way more.

ALso you may cleave the tag later if it interferes with protein function. Bacterial expression systems like 6xhis, eucaryotic ones prefer myc. But as Phoebe wrote :"it depends" - and not only about expression system but also on the downstream applications of your protein of interest.

I will also have to say "it depends". Creating and testing both N-terminal and C-terminal is honestly your best bet. In one system, I had one version work fine and the other caused E. coli to form slimy masses that only grew in terrific broth (and poorly at that)--the HIS tag closed off access to a functional binding cleft. I also agree with Krzysztof Wicher: knowing more about the functional domains may help you make a more educated decision about where to place the tag. If it is not known, try both! Phoebe Stavride also makes a great point about being careful--know exactly what your vector will be inserting into your protein!

It depends on your protein I would say too. Our experience is that certain GPCRs show a dramatically decreased surface expression when tagged at the N-terminus, while others are well expressed, but show an altered signalling upon c-terminal tagging. So as my forewriters suggested - try both and compare carefully against untagged controls in case you do functional studies with the tagged protein. What do you need the tags for? Purification or detection?

go N-terminal, should be fine! or test C-ter as well just to be sure, but in the middle..I would't start with that

best & good luck

It simply depends of the protein

I would suggest you adding FLAG tag just after the start codon of endogeneous gene you are interested in in human somatic cell line, because whether you add the tag to N- or C- terminus of the gene in an expressing vector, the protein encoded by the gene all will not be expressed naturally, futher will affect its folding and function.

How would you do this? I mean modifying the genomic sequence to carry the flag tag?

The His-tag allows to purify proteins in denaturing conditions if they are expressed in inclusion bodies of E.coli. If you are going to add tag sequences to any protein I suggest to copy them from commercial plasmids because the codon usage may be different if you express proteins in eucaryotes or prokaryotes

Hi everybody, all of you are discussing about His tagged fusion protein I would like to ask a question. I just finish my his tag fusion protein and hoping that this protein will be soluble as I need it in native state so that I will be able to do stuctural study (for secondary stucture and metal binding study).Unfortunately all of the protein was found in inclusion bodies and almost non in supernatant after lysis. Any ideas from you guys. last time I tried with GST tag with the same protein, I manage to get good soluble protein but the end product was too low (due to binding capacity of GST column) for me to do any structural/ crystal study. Thanks

There is a number of things you can do to try to make your protein soluble. Decreasing temperature and/or IPTG concentration should make the production slower and more tolerable to the bacteria (you will need to increase the incubation time to get enough protein in this case). Increasing air can also help (make your flask lids really loose, use bigger flasks/lower volume). Let me see if I can find a suitable link for you (and perhaps the original poster).

Yes, here it is:

http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/LD_149682128-R350?OpenDocument&hometitle=tech_support_service&ParentDoc=A5398443586C5EA7C125763B00328FDC

You can find more handbooks (including one for GST proteins specifically) here: http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/service_and_support~documents_and_downloads~handbooks

I strongly recommend to all to check the list - there are some really good handbooks in there and the pdf versions are free.

Before to screen the hundreds purification protocols I would suggest to Hasnah to run a hydrophilic-hydrophoic plot of your protein to see if you can express it in fragments or functional domains which could be still useful for an a comprehensive crystal work

Ok let me clarify. My first post gives a specific answer to a specific question. If you want to make your protein soluble, try making the changes I suggested during the expression step. Might work, or you might lose a day.

My second post gives a link to more background for those interested, be it Hasnah or the original poster, since personally I prefer understanding and deciding for myself, rather than randomly following conflicting advice. It probably also contains a more complete troubleshooting table than the few basic things I wrote on my first post and in a hurry, but you definitely won't see it writing anything different.

So, if you don't like reading the background stuff, by all means, just try what I said on the first post - you don't have to bother with the second post at all.

I agree with the ideas here, that it really dependes on the protein. Moreover I am not a big fan of Myc and his tags (unless it is bacterialy expressed for His tag), The Myc is very large and can affect the function/stability of your protein, His antibodies I don''t like. I personaly use HA and Flag either N-terminus or C-. you can add a small (4-8 amino acid linker if you wish).

But like all in Biology hope for some luck

P

The topic arouses great interest, I am happy and thanks to all.

I apologize with Phoebe, I did not want to minimize your comprehensive answers. I would only give a methodological starting point for younger people.

Before to start your work of protein expression I suggest to make prevision answering these questions:

1) Was the protein already expressed or does it resemble to any protein already expressed?

2) Has the protein any activity that could be deleterious for the cell recipient ?

3) Do I know everything that is possible to know of my protein running its nucleotide/protein sequences on bioinformatics programs available such as http://www.expasy.org/ ?

Fortune needs some help!

Dear Paola, all is good :)

Hasnah, I just realised you said metal binding. If your protein has a metal atom in it, then it is really important that you check if your protein or similar has been previously expressed, as Paola suggested, for there may be special requirements. For example, many buffers use EDTA which can chelate many ions and remove them from your buffer, which doesn't exactly help your protein fold properly... better check a few papers that deal with your protein or a family member of it, to see what kind of protocols they use.

Thanks Phoebe and Poala for the comments. I will analysed the metal content with ICP MASS, but for metal binding affinity I will be doing Fluorescence study and CD. Currently I do not have enough protein as the His tag cconstruct was the 2nd choice after GST as I found GST caused my protein percipitated and digested on the column. When I purified my protein in the presence of EDTA, ICP MS analysis showed 1 zinc per monomer present in my protein. This protein was predicted to bind Mn2+. Probably Zn bind to protein for stability of the stucture. During exressio

Hasnah, break you cells in the presence of 0.2% triton X-100 or last resort is gradual increase of urea up to 2M.