Guys, I have a pET vector 30a+ and my gene of interest has been inserted between XhoI and KpnI sites. Nearest to it is only an S-tag present. How can I purify my protein and remove the s-tag from it.
I'm attaching the plasmid map along with it to visualize a clear picture.
After looking at the vector map and the enzymes used for inserting your gene of interest, I can suggest you go for His-affinity(Ni-NTA) purification as it will be more economical, and also after purification, you can remove the His-tag by thrombin cleavage to get your protein of interest.
But if you want to do S-tag purification then you cannot separate the tag anyhow as there is no cleavage site in between your protein and the tag.
I am attaching a file that might help you in S-tag purification.
Hi,
There is one enterokinase cleavage site between you protein and the S-tag. You can do Ni-NTA purification and remove the N-terminal His6-S-tag. Alternatively, if you dont mind the C-terminal His tag, you can insert you gene in NdeI/XhoI, that will leave you a C-ter His6 tag. I hope this helps.
Hi!
I have checked it again AMIT KUMAR GAURAV , but the enterokinase site is located after the KpnI restriction site and has been eliminated because of the insertion of my gene of interest.
- Badges
- Science topic