 |
 |
Hi Eugenio, It will depend on the expressing system you are using: mamalian cells, insect cells, yeast. For enzymatic deglycosylation I suggest you to do first do a small scale test, using the following enzymes: PNGase F is extremely efficient. EndoH (you can try EndoHf which is cheaper and ) Neuraminidase to remove the sialic acid (specially useful for mammalian cells). You can check these enzymes at NEB or Prozyme (like Radu mentioned before. Check also his great paper Chang et al, 2007.). http://www.neb.com/nebecomm/default.asp You can also use glycosylation inhibitors like tunicamycin, kifunensine, swainsonine, etc... Another approach is site-directed mutagenesis. And yes, "usually" (this means not always...) it gives you better crystals which will diffract at higher resolution. Good luck, Joao Joćo M. Dias, Ph. D. Ollmann Saphire Lab The Scripps Research Institute 10550 North Torrey Pines Rd. IMM-2 La Jolla, CA 92037 USA tel (858)784-8925 http://www.scripps.edu/~jmdias/ On Sep 25, 2008, at 1:40 PM, A. Radu Aricescu wrote: > Dear Eugenio, > > I believe the paper by Chang VT et al 2007 (PMID: 17355862) might > offer the answers you're after (assuming N-glycosylation is your > main concern). For O-linked sugars, the situation is much more > complex, reflecting their diversity: mucin-type can usually be > dealt with by construct design (eliminate Ser/Thr/Pro-rich > domains), for other types have a look at http://www.prozyme.com/pdf/ > gk80110.pdf for example. > > Best wishes, > > radu > > > ------------------------------------------ > A. Radu Aricescu, PhD > University of Oxford > Wellcome Trust Centre for Human Genetics > Division of Structural Biology > Roosevelt Drive, Oxford OX3 7BN > United Kingdom > Phone: +44-1865-287551 > Fax: +44-1865-287547 > > > ---- Original message ---- >> Date: Thu, 25 Sep 2008 11:30:24 -0700 >> From: Eugenio De la Mora <[log in to unmask]> >> Subject: [ccp4bb] Deglycosylation >> To: [log in to unmask] >> >> Dear all, >> We have some troubles with the cristallization of glycosylated >> proteins and want to try to deglycosate them. We have never done >> this so we want to know what enzymes are the best (efficiency; >> less protein loss, ... ). And if it gaves better results in >> crystallization screens. >> >> Thank you >> >> Eugenio De la Mora >> Instituto de Biotecnologia >> Universidad NAcional Autonoma de Mexico >> >> >> >