Dear Simon, this may be an isolated case, but you might want to try to drastically change the pH of the reaction conditions. In our hands, a protein with 3 hyperglycosylated sites, expressed in Pichia pastoris, could be deglycosylated readily with endoH. However, at the recommended reaction pH (5-5.5), the protein rapidly precipitated in an irreversible manner. After testing the reaction in various different buffers, nearly all of the protein could be deglycosylated at pH 7.5, with no visible precipitation. Curiously, the buffer could easily be exchanged to pH 5.5 and below after the reaction was finished (the protein even crystallized at pH 4).
This of course only has a small chance of working in your case, but it's quick to test various different mini-deglycosylation experiments using an array of conditions. Cheers Filip Van Petegem On Mon, Mar 16, 2009 at 9:29 AM, Yue Li <simon.yu...@yahoo.com> wrote: > Hi everyone, > > Recently, I obtained a soluble glyco-protein. Unfortunately, after I added > PNGase or Endo Hf to remove the glycans, the deglycosylated protein is > precipitated. Is there any method to avoid this kind of precipitation? > > Thanks, > > Simon > > -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
