pET-28a(+) vector contain few fusion tags. N-terminal His.Tag and N-terminal T7.Tag along with C-terminal His.Tag.
What are the individual usage of each tags?
If all tags were there with my insert will it be a problem for expression of native protein?
Why C-terminal tag code for 'pro' instead of 'His' and benefit of each?
Map of the vector: file:///C:/Users/hp/Downloads/TB074VM.pdf
Thank you.
Hi Nilmini,
pET28 is designed so that you can clone your gene in to have a choice of N-, C- or no tags depending upon which cloning sites are used. So you can generate constructs such as:
Start-HisTag-GOI-Stop, Start-HisTag-T7Tag-GOI-Stop, Start-GOI-HisTag-Stop, Start-GOI-Stop. You can also make Start-HisTag-T7Tag-GOI-HisTag-Stop but this is probably not that useful.
The His tag and the T7Tag can be used used for both detection and purification. The His tag is probably the more commonly used of the two.
For the C-terminal tag there are three vectors a,b & c each in a different frame so that you can clone your gene into the right frame to generate the His Tag, the Prolines you see in pET28(a) are simply an artefact as they are out of frame with the rest of the tags.
If you wish for a totally native protein you must express it without tags, however cloning at the NdeI to one of the other sites (say BamHI) and including a stop before the BamHI would give you a Start-HisTag-GOI-Stop construct that has a Thrombin cleavage site, so purification on IMAC is possible. Subsequent cleavage with thrombin would leave only a GlySerHis before the Met start. In many (but not all) cases this can be functionally the same as the native protein.
Whether a N- or C- terminal tag is best depends upon your protein and your end requirements. If you don't have prior knowledge as to which to use it is best to construct both N-taged and C-taged proteins and decide later which is best for your needs.
-Richard
Dear Nilimni Kumari: There is an issue of tag being buried into the fused protein and that is why you have the choice of selection. Some fusion proteins will express the His-Tag at the C terminus and some on the N-terminus. If you express the tag at the N terminus then you can make truncation at C terminus and all the deleted proteins will be easy to isolate for any study.
Hi Nilmini,
pET28 is designed so that you can clone your gene in to have a choice of N-, C- or no tags depending upon which cloning sites are used. So you can generate constructs such as:
Start-HisTag-GOI-Stop, Start-HisTag-T7Tag-GOI-Stop, Start-GOI-HisTag-Stop, Start-GOI-Stop. You can also make Start-HisTag-T7Tag-GOI-HisTag-Stop but this is probably not that useful.
The His tag and the T7Tag can be used used for both detection and purification. The His tag is probably the more commonly used of the two.
For the C-terminal tag there are three vectors a,b & c each in a different frame so that you can clone your gene into the right frame to generate the His Tag, the Prolines you see in pET28(a) are simply an artefact as they are out of frame with the rest of the tags.
If you wish for a totally native protein you must express it without tags, however cloning at the NdeI to one of the other sites (say BamHI) and including a stop before the BamHI would give you a Start-HisTag-GOI-Stop construct that has a Thrombin cleavage site, so purification on IMAC is possible. Subsequent cleavage with thrombin would leave only a GlySerHis before the Met start. In many (but not all) cases this can be functionally the same as the native protein.
Whether a N- or C- terminal tag is best depends upon your protein and your end requirements. If you don't have prior knowledge as to which to use it is best to construct both N-taged and C-taged proteins and decide later which is best for your needs.
-Richard
Dear SIr Richard and Sir Ishtiaq,
N-terminal 12..70 amino acids region of my GOI act as a CPx-type heavy metal Associated domain, and it is a conserved region. If so N-terminal tags may influence to the native protein function, isn't it? And the C-terminal domain 625...707 residues are code for HAD-like domain. Total length of the protein is 795 amino acids. what would you think ? Which way will be better?
I am glad to here from you.
Hi Nilmini,
it's difficult to say which (if either) would be better. You really need to try both to see which works for you. The 12 amino acids before the N-terminal domain could well be sufficient, it depends upon the protein's structure. Again depending upon the structure there is a fairly good chance that the C-terminal will work. The best option is always to generate different constructs and test them out.
I suggest that you check all the literature you can to see if anyone has had success with your or similar proteins in E.coli. Any structural information you can find would be very useful.
You have quite a large protein there, so you might also have to optimise your expression conditions to get good expression.
-Richard
Dear Nilmini,The tags will allow you to detect and purify your protein using a nickel or a Cobalt column, for example. But if you want a native protein, your protein needs to be express without Tag, you can use the restriction enzymes specific for your vector. Some researchers prefer using His-tag in C-terminal when use this construct for animal immunization.
The fate of your protein might affect your choice of tag position: Sometimes, proteins contain signal peptides (frequently at the N terminus). If you add a tag there, the location / fate of your expressed protein will/may be "incorrect".
Ok, that means for the determination of function of the GOI does not need His.tag at their terminals but for the purification purpose only. If I want both better to have two different construct, with Tags and without Tags, am I correct?
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression