Dear Jerry,
First of all, it will be hard to reproduce the conditions with the glycosylated protein because by its nature, it is heterogeneous. One thing I would try with the glycosylated protein is a detergent screen, or if you don't have one, use a few NDSB's. Second, I would try setting up a lower PEG condition concentration and seed into it with the phase separated material, or with polystyrene nanobeads. Third, I would try FID (Free Interface Diffusion- like Microlytic's Crystal Former- www.microlytic.com) experiments, or use Enrico Sturza's technique with the Granada (?) Crystallization Boxes. Have you tried shifts in temperature? Start by warming up the protein to between 37-42C, then set up the drops. Allow the plate to equilibrate at some temperature between 37-20C then transfer to 4C after 24H. Something I suggest is that you go back to construct design and mutate the amino acids being glycosylated to something like GLY or ALA. If you can, express the protein in a different system (if you used Ecoli, try Insect cells or vice-versa). Alternately, you can use SGC's method of dilute protease mixed with your protein prior to setting up the drops. I have tried 1:10000 dilution, but I think that is too weak. I would start with 1:100 or so. Good Luck, and let us know how it turns out. Regards, Bryan Prince. From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Jerry McCully Sent: Wednesday, March 03, 2010 10:59 AM To: [email protected] Subject: [ccp4bb] crystallization of highly-glycosylated protein complex-avoid phase separation 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. <http://clk.atdmt.com/GBL/go/201469230/direct/01/> -------------------------------------------------------------------------- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
