There are many cases in literature where the removal of N-linked glycosylations sites reduced the expression levels of secreted proteins. I would recommend removal of the N-linked glycans by enzymes (endo H, PNGase F, etc.) using the wild-type proteins you have obtained and then run a gel or mass spec to determine the M.W. after deglycosylation. Alternatively, you may just leave the glycans on if the mass from glycans are just several thousand daltons (how large is your protein?). Many crystal structures of secreted proteins are solved with the glycans on. They seem to help protein folding in your case.Holly Jing
> Date: Tue, 4 Mar 2008 16:54:00 -0500> From: [EMAIL PROTECTED]> Subject: Re: > [ccp4bb] Removal of glycosylation sites in Picha expression construct> To: > CCP4BB@JISCMAIL.AC.UK> > This is not entirely uncommon. Did you try removing > just one of the two> sites (sometimes it helps) - combined with enzymatic > deglycosylation this> may give you good enough protein to work with.> > If > you try other hosts, I would definitely consider Schisosaccharomyces> pombe - > its glycosylation patterns are much simpler. Other likely> candidates include > regular bakers' yeast and Hansenula polymorpha. I would> try both the native > and the mutant proteins in those hosts.> > There are tricks one can play with > Pichia to try and make it express the> deglycosylated protein but it almost > sounds like you've hit on something> very essential since you're going from > good expression down to no> expression.> > Lastly, you could try insect cells > :)> > Artem> > >> > Dear all,> >> > Our lab is new to working with Pichia > pastoris, also new to working with> > glycosylated proteins. We have a > construct for a secreted protein that> > expresses pretty well in Picha, but > upon mutation of the 2 N-linked> > glycosylation sites to Ala, we get no > expression at all, nada. The> > nucleic acid sequence appears to be correct, > i.e. we have not introduced> > any unintentional frame shifts, stop codons, > or anything like that. Is> > this a common phenomenon? Are there any tricks > to get the Pichia to do> > its thing? Any chance that alternative > substitutions will work when Ala> > does not? Or are we better off (a) trying > to deglycosylate> > enzymatically, or (b) trying a different expression host? > All opinions> > and anecdotes welcome.> >> > Thanks!> > Evette> >> > Evette > S. Radisky, Ph.D.> > Assistant Professor and Associate Consultant II> > Mayo > Clinic Cancer Center> > Griffin Cancer Research Building, Rm 310> > 4500 San > Pablo Road> > Jacksonville, FL 32224> > (904) 953-6372 (office)> > (904) > 953-0046 (lab)> >> > _________________________________________________________________ Connect and share in new ways with Windows Live. http://www.windowslive.com/share.html?ocid=TXT_TAGHM_Wave2_sharelife_012008
