Hi Purva

How is your matrix?  A bacterial cultural media, a solution of a cell-disruption?

Natural protein or GMO?

If it is possible to add a his-tag to a cloned protein, you will have a bigger chance for a quick clean-up

Ultrafiltration will be the first choice to isolate a near range of your desired molecules, but probably will not give you a real sharp cut. if you want to avoid SEC, an ion-Exchange resin might be a good alternative to distinguish between PEGylated and non-modified fractions.

Thank you sir for your response.

I am working on purified uricase, so doesn't use any culture media. Instead, i use SPB as reaction buffer for PEGylation of protein. Affinity chromatography ( Xanthine agarose) and CM cellulose for ion exchange chromatography, i was also thinking about. Thank you for that.

Can i use ultrafiltration as protein fractionation method, to separate protein and its conjugates?  the retentate recovery will be very low?

Hello,

I am afraid that your difference in MW is not enough to use ultrafiltration! Also I would not use affinity for uricase, as it may not distinguish between the free and the PEG-bound enzyme.

I would rather suggest ion exchange or manual SEC. It's not as easy as the automated one, but should be fine!

Ultrafiltration is unlikely to be useful for separating the forms of the protein. Hydrophobic interaction chromatography, such as with phenyl-Sepharose may be useful in this situation. I would think that PEGylation would have a substantial effect on the salt-dependent binding of the protein to the column. Another method that might work is ammonium sulfate fractionation, which might separate the forms on the basis of precipitating at different ammonium sulfate concentrations.

Thank you so much for your response. I'll look forward to it.  I havn't observed any physicochemical property changes yet except increase in molecular wt. , since, hardly one or two PEG binds to the protein,since, i am following a second generation PEGylation technique. I will try to find out other changes taking place.

I suggest you read our publications on PEG-uricase, such as Sherman, M. R., Saifer, M. G. P., & Perez-Ruiz, F. (2008). PEG-uricase in the management of treatment-resistant gout and hyperuricemia. Adv Drug Deliv Rev, 60, 59-68. If you send me an email, I'll send you several reprints.  [email protected]