Jerry, One thing you might try is a combination of Endo F1, F3, and possibly Endo H(might trim a little bit more). This works for me on glycoproteins expressed in insect cells which show little or no sensitivity to PNGaseF and EndoH. Depending on the host strain you are using to express, the sensitivity to different glycosidases will change. Other approaches are to add kifunensin to the culture media, which inhibits mannosidase trimming, resulting in high-mannose carbohydrate that will be sensitive to PNGaseF and EndoH. However, one might assume that the protein will need some amount of carbohydrate to be happy, so that may not improve you crystallization (for one project the best crystals ended up being fully glycosylated). In one instance removing a single carbohydrate (1 out of 5 on a 50 kD human glycoprotein) though point-mutagenesis created a crystal contact at that very site which allowed me to solve the structure. Enzymatic deglycosylation with EndoF1 and F3 did not produce good crystals.
If it is a mammalian expressed protien, you can use the EDEGLY kit from sigma to sequentially remove the terminal sialic acids, beta-galactose, and beta N-acetyl glucoses, at which point it should be PNGaseF sensitive. Or, you could use a mannosidase to get back to a glucNac_2 carbohydrate. We have had better success with material produced in insect cells, as they only put on pauci-mannose carbohydrates, which are much smaller and not charged like mammalian carbohydrates. In fact we have seen those glycans participate in favorable crystal contacts that would not be possible with mammalian glycans. So, there are ways to deglycosylate it and change the glycosylation, or course it's not guaranteed to improve the crystals, but it's something to try. Hope that helps, Nat On Wed, Mar 3, 2010 at 10:59 AM, Jerry McCully <for-crystallizai...@hotmail.com> wrote: > Dear ALL: > > Recently we've been trying hard to crystallize a highly glycosylated > protein complex ( 30% percent of carbohydrate in the total 120KD molecular > weight). > > IT is a high affinity protein complex. One component can be crystallized > in high salt condition and the other can be crystallized in PEG. > > Through screening, we happened to get some very ugly crystals in PEG > condition using gel-fil purified complex sample. > > The problem is that it is very hard to repeat the screening condition. In > contrast, it is very > easy to get phase separation in the drop although we tried to optimize the > protein and precipitant concentration. > > We tried to de-glycosylated using PNGase and EndoH but failed in > non-denaturing condition. > > Hampton additive screen did not help either. We tried seeding several times > but so far we have not got any better luck. > > Can anyone give some guidance here? Thanks a lot, > > Jerry McCully > > > > > > ________________________________ > Hotmail: Powerful Free email with security by Microsoft. Get it now.
