I have purified a recombinant protein with a His tag(no cleavage site ) for oral administration in mice (via orogastrically) . Is it necessary to remove the His-tag before invivo studies??
If so , what are the possible strategies to remove it ??
While, it is not strictly necessary, it might depend on antigen of interest and experiment in general.
I don't think it is necessary to remove the His-tag, in general. If you want to remove it, you should use a construct that contains a specific protease cleavage site between the His tag and the protein, such as a TEV or thrombin site. After cleaving off the tag using a His-tagged protease, the material is passed over an IMAC column to remove the tag and protease.
A protein administered via the oral-gastric route will most likely be digested by proteolytic enzymes in the gut and destroyed..
Dear Shwetali Naresh Prajapati
it depends from the aim of your experiment.
For example if you are mmunizing animals with a recombinant antigen contaning an his-tag for antibody generation the presence of his-tag may induce some antigen non specific anti-his antibodies and it have to be considered when you are desing the elisa screening to select antigen binding clones.
Removal of his tag is generally performed by adding during the cloning phase the sequence codifing for a protease as TEV or enterokinase or Fatt.Xa between the his-tag and the antigen sequence, which allow to perform his-tag removal after first step of imac purification.
The digested Histag could be then removed from the protein with a SEC or with a subctractive imac step
you can find more information about subctractive imac on the following video
https://www.blogger.com/video.g?token=AD6v5dxhCeJUPx5UODBu4Y9hhJZix_l5JZcPb03ZUe7G3qL8GhoPUWZMm6z2wEJJED9OY8gn_ttNXrGNCdVurZ6zkB_oTSCrmRc90-ux7AQAOhaddCkXKREYmgNw87RSh58zQ4uVQVtz
present on my blog: ProteoCool
good luck
Manuele
Manuele Martinelli, Adam B Shapiro , Darshan Panchariya I plan to use a recombinant His-tagged protein for orogastric administration in mice to study its adhesion to the gut lining. Is it necessary to remove the His tag for this type of in vivo study?
If you want to see the effect of your protein only then it would be better to design the protein accordingly before starting any in vivo experiments which could become worthless in the worst case if the His-tag does indeed change the properties of your protein (could have false positive or false negative result). At least you would avoid the same questions that you have now from reviewers when you submit your study with the His-tagged protein for publication.
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