Lectin affinity chromatography resins can be used to purify glycoproteins based on an interaction with specific sugars. The protein can be eluted using the sugar that is recognized by the lectin.

Dear Homasadat Esfahani

the problem is that if a protein is higly glicosilated its surface is masked by sugar molecules and the real pI of the protein could be really different from the theoretical. This do not means that ionic exchange could be not applied but that you have to perform some trials with several pH for the bindings.

As Adam B Shapiro suggest, you can also exploit the presence of sugars to use some resin that specifically bind it as for example

https://www.takarabio.com/documents/User%20Manual/PT4050/PT4050-1.pdf

but i suspect that in this way you will purify also other proteins that are glycosilated from Pichia and you will not realy enrich it.

I suggest you to add an N- or C-terminal histidine tag cleavable with a proteases as TEV for example, that could be removed after purification and in case use SEC chromatography or subctractive IMAC as second step.

best

Manuele

Try concanavaline A resin which is highly succesfull for capturing glycoproreins...Jacalin may also works well..Lentil lectin is the most predominantly used..you should packed your own column or must use them as gravity column or as batch mode

Manuele Martinelli Thank you so much for your advice. Indeed, my problem is that my target protein is hyperglycosylated; therefore, I can't capture it by the columns.

Adam B Shapiro and İsmail Emir Akyildiz Thank you so much. unfortunately I don't have an access to the lectin resin.

• If it does not have any tag and it is not possible to add to your experiment
• if you cannot apply lectin isolation,
• If only global protein purification resins in your hand to implement in your workflow, (IEX, HIC, SEC, Mix-mode, etc)
• If any IEX does not work (it does not make sense much, as indicated previously working around the pI of the POI caused by its highly glycosylated nature should solve the issue. Maybe AEX reflects some insights since oligosaccharides attached to your protein bring a major negative charge to it),
• If it does not bind to any resin,

Then maybe the flow-through strategy is the way to choose.

Tandem purification using AEX-CEX-HIC-SEC that is applied to every flow-through fraction sequentially may do the job. Polishing-based strategy (wash-out mode) instead of bind-elute mode does not perfectly but satisfactorily can purify your protein target.

Good luck...

Emir

İsmail Emir Akyildiz Many thanks for your help.