I have cloned a gene in pET28a with a protein size of 47KDa, I am obtaining fairly good yield after induction and purification using the Ni-NTA Agarose (Qiagen). I would like to know the easiest and a most rapid method of His Tag removal. I don't think Thrombin can be of help since its slow and cleaves randomly.
Please help.
Thrombin should be specific for Leu-Val-Pro-Arg-Gly-Ser. If you are getting nonspecific protease activity, the purity of the thrombin may be an issue. This lab cleans up their thrombin with a Mono-Q ion exchange chromatography step:
http://structbio.vanderbilt.edu/chazin/wisdom/labpro/thrombin.html
Double check your protein to find out which cleavage sites it has before selecting a cleavage sequence for your tag.
If you don't want to use Thrombin, try AcTEV or PreScission. They've worked well for me. You might try Clontech's inFusion cloning system to change the cleavage site in your vector if you can't find His tag vectors with the cleavage sites you want.
Why to remove HIS tag..you can further proceed your protein for some experiment if its not interfering in any way then no need to remove HIS tag.In our lab we frequently use PET series and PQE-30 vector which has HIS tag, we never remove the HIS tag and we never found any interference of HIS tag with any type of activity or interaction studies..Think abt it.
you can use enterokinase to remove his tag. but you should clone thioredoxin seq after your N terminal-his tag .
The easiest way is to use TEV protease (very specific) which also has a HIS tag itself. After cleavage (30C 4h or 4C overnight) you can run another Ni-NTA column, but this time, collect the flow-through, because the column will bind the cleaved tag and TEV protease itself. For us it works perfectly fine.
We also express our own home-made TEV protease with HIS-tag. So clone the TEV protease into a pMAL or pGEX plasmid and you can make high quality protease of your own for cheap.
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