Hi,
Is there anyone who know about glycosylation of protein could inhibit His6 tagged-protein binding to Ni-NTA resin?
I was trying to purify secreted protein from pichia using Ni-NTA resin and I confirmed the new band in my mutants (no band in control). However, this protein does not bind to Ni-NTA resin.
I just want to know is there any reports about inhibition effect to binding of glycoslyated protein.
The original protein size is 50 kDa and glycosylated protein size is 70 kDa.
Mohamed,
I know there's some common problem with His6. But, in my case, it is successfully purified when I purified my protein intracellular form. So, I don't think common His6 or domain issule is not the problem. Just I wonder, if the glycan could inhibit binding of His6 tag to Ni-NTA resin.
Anyway, Thanks for your comment.
Hi Jun-Seoh,
Glycosylation has been found to alter the secondary and tertiary structures of proteins so it certainly could be a factor in your case. The presentation of the His tag could be altered by glycosylation, resulting in reduced binding affinity to the resin and thus reducing your yield.
Hi, maybe you could help to block sugar hydroxyl groups with acetylation before IMAC, but I dont know if it is possible due the denaturation of your protein.
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