Hi all
I am interesting if you want to produce some protein and have plasmid, the way is to grow it in bacteria and than isolate with antibody which will bind tag (example FLAG tag etc.) but what to do if you dont want have tagged protein at the end ? should remove tag? or is better dont use tag? than what is the best strategy to purify it?
Dear David,
if you want to get rid of the tag, you can e.g. introduce a protease cleavage site into your plasmid sequence. Then you can cleave the tag after protein purification or even better when the protein is still bound to the affinity matrix. For many applications a tag does not do any harm, but you have to check carefully whether the tag modifies the function of your protein. However, often it is nice ta have a tag when you do not have specific antibodies for your protein of interest.
You can also use a system exploiting the self-cleavage activity of intein to purify untagged protein ( search for Intein Mediated Purification with an Affinity Chitin-binding Tag). Hope this helps a little.
Best regards,
Moni
Dear David,
if you want to get rid of the tag, you can e.g. introduce a protease cleavage site into your plasmid sequence. Then you can cleave the tag after protein purification or even better when the protein is still bound to the affinity matrix. For many applications a tag does not do any harm, but you have to check carefully whether the tag modifies the function of your protein. However, often it is nice ta have a tag when you do not have specific antibodies for your protein of interest.
You can also use a system exploiting the self-cleavage activity of intein to purify untagged protein ( search for Intein Mediated Purification with an Affinity Chitin-binding Tag). Hope this helps a little.
Best regards,
Moni
I agree with Monika, above. Here's a really good overview of various affinity tags you can use for protein purification: Article Current Protocols in Protein Science
Our lab typically makes use of hexa- or octa- Histidine tags, which you can purify using Ni-NTA or Cobalt resin column chromatography. We typically place an enterokinase cleavage sequence (https://www.neb.com/products/p8070-enterokinase-light-chain#Product%20Information) between our His-tag and our protein of interest, and then cleave it after purification. You can then use simple size-exclusion (such as an Amicon centrifugal filtration tube) to separate the cleaved tag from the protein of interest. Hope that helps! Good luck!
Usually when you want to remove a tag attached to your protein, in the plasmid while cloning , you clone a TEV protease site between the protein gene and your tag gene. After elution of the protein due to the tag, you add tev protease at a ratio of target protein/tev protease = 50/1 and dialyse it to remove what ever elute buffer you had before. How pass this digested protein and collect the flow through to remove any protein still not digested by tev protease as it will stick through the column and digested wont.
Hi David,
I would recommend to follow Monika Stefanie suggestions. We also purify protein using His-tag, which we have to cleave at the end or before solving its structure. We simply perform cleavage using TEV protease sometimes in 10/1 (protein/ TEV) or sometimes also increase this ratio to 20/1.
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