(HELP)Properties of protein after glycosylation - any effect on pI n etc? (May/17/2006 )
Hi,
i am looking at a protein which is highly glycosylated. would like to know if the glycosylation affects any other properties of the protein, for eg, the pI. currently expasy ony notes/calculates the properties of the protein without glycosylation. can anyone pls help?
thank you
Glycosylation affects solubility and stability as well as functional characteristics. Sialic acid residues on antobody molecules gradually get knocked off to reveal the naked protein which is recognised and degraded.
There will be hydroxy groups on the sugar residues that could easily give up a hydrogen which would affect pI..but would this occur at the pH you are working at? It may be that at physiological pH there is no effect and the pH required to bring about disruption at the sugars would grossly affect the protein itself.
You could try a series of experiments using glycosidases. Run a gel (doubt you'd see much of a shift) try HPLC (same, little effect) try light scattering, dialysis and solubility.
One thing, glycosylation is often used to get the protien out of the cell to do its job. The sugars may be part of receptor binding sites so I think you'd have to express the intact protein then remove the sugars rather than prevent glycsylation in the first place. Just how you check that you've removed all the sugar may be hard.
There will be hydroxy groups on the sugar residues that could easily give up a hydrogen which would affect pI..but would this occur at the pH you are working at? It may be that at physiological pH there is no effect and the pH required to bring about disruption at the sugars would grossly affect the protein itself.
You could try a series of experiments using glycosidases. Run a gel (doubt you'd see much of a shift) try HPLC (same, little effect) try light scattering, dialysis and solubility.
One thing, glycosylation is often used to get the protien out of the cell to do its job. The sugars may be part of receptor binding sites so I think you'd have to express the intact protein then remove the sugars rather than prevent glycsylation in the first place. Just how you check that you've removed all the sugar may be hard.
thank you for your lenghty explanation. i guess tht the best way is to allow the glycosylation to occur and then remove it. i am looking at using PNGase F to remove the glycosylation so tht i am able to detect it due to the fact tht my protein is in the 55kDa to 75kDa and it is hard to detect/distinguish. any other soft pufiying method tht i can use to purfiy a protein which i do not know the pI value and other properties? immunoaffinity seems possible rite?