I have a clone of 9kDa protein in pET28a, at EcoRI and HindIII. Can anyone suggest me, how can I remove the His tag?
Hell Avijit,
there is a little bit more information necessary:
a) do you want to remove the Tag after protein production? than you should insert a Protease cleavage site (e.g. TEV) between your protein of interest and the tag (via Primer design)
b) do you want to have an untagged protein?
than you need to consider this to get rid of the tag option during cloning, by choosing the right enzyme/designing appropriate primer, so that the tag ist not translated..
Best regards,
Pascal
Hi there,
By looking at pET28 map , your construct would yield a protein with His tag at the Nter and also a his tag at the Cter if you have not included a stop codon on the HindIII side of your insert. There is a TEV cleavage site between the Nter His tag and the EcoRI site therefore you can remove this tag by TEV protease treatment. A good way to get rid of the TEV and of the digested tag is to repass the digest onto NiNTA (provided the TEV protease is itself His tagged). The untagged protein (if not Cter his tagged...) will pass through whereas tag and TEV will be retained.
pET28a has a thrombin site so use thrombin to cleave the tag.
You're better off in the long run using a modified pET28 vector with a TEV protease site. You can easily make your own His6 tagged TEV so that post digestion you can easily remove both the cleaved tag and the tagged-TEV in one step using Ni-NTA.
Thank you Prof. Dominique Liger
Thank you very much.
Can you please reply me the sequence, which is coding for TEV restriction site( E-Xaa-Xaa-Y -Xaa-Q-(G/S), in between N-ter His-tag and EcoRI sites.?
Hi,
the sequence recognized by the TEV protease is: ENLYFQ\S cutting between Q and S.
Best,
Piero
If you're going to make your own TEV I suggest getting the plasmid form this reference: PMCID: PMC2047602
Plasmid is available here: http://dnasu.org/DNASU/GetVectorDetail.do?vectorid=411
Yield is over 100 mg per liter and it has a His6 tag for easy purification.
Hi,
Just a small addition to the protease restriction site.
This site is very helpful:
https://www.oxfordgenetics.com/SiteContent/TeamResources/cleavage-tag-guide
Kind regards,
Simon
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning