Dear Alisa, Thank you for your suggestions. The carbohydrate should be N-linked and the protein was expressed in HEK293 cells. Chances of getting another protein batch in the short-term are slim, so I am looking for ways to trim existing carbohydrate chains. I will look into PNGase F.
Best, Herman Von: Alisa Glukhova [mailto:alis...@umich.edu] Gesendet: Mittwoch, 15. Oktober 2014 15:37 An: Schreuder, Herman R&D/DE Cc: CCP4BB@jiscmail.ac.uk Betreff: Re: [ccp4bb] Trimming of carbohydrate chains Dear Herman, You did not specify the site of glycan attachment (is it N- or O-lonked?). I assume it is N-linked as your carbohydrate is quite large. And what expression system are you using? In general you can first try PNGase F, however if you are unlucky it will only cleave in the presence of SDS. Other strategies include either using the inhibitor of glycan pathways e.g. kifunensine or swainsonine (Chang, V. T., Crispin, M., Aricescu, A. R., Harvey, D. J., Nettleship, J. E., Fennelly, J. A., et al. (2007). Glycoprotein structural genomics: solving the glycosylation problem. Structure, 15(3), 267–273. ). or expression in different systems - i.e insect cells instead of mammalian cell culture. You could also look into glycan deficient cell lines such as HEK293S GnTi- or CHO lec 3.2.8.1 (Reeves, P. J., Callewaert, N., Contreras, R., & Khorana, H. G. (2002). Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proceedings of the National Academy of Sciences of the United States of America, 99(21), 13419–13424; Stanley, P. (1989). Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity. Molecular and Cellular Biology). Best, Alisa On Wed, Oct 15, 2014 at 9:03 AM, <herman.schreu...@sanofi.com<mailto:herman.schreu...@sanofi.com>> wrote: Dear Bulletin Board, I am struggling with a protein domain of 15 kDa, with about 22 kDa carbohydrate attached. So far, the domain did not crystallize and I suspect the carbohydrate may hinder crystallization. Completely removing the carbohydrate results in low expression yields and poorly soluble protein, so I would like to try to remove some, but not all carbohydrate. Does anyone has a good protocol to trim, but not completely remove the carbohydrate? Thank you, Herman Schreuder -- Alisa Glukhova graduate student Tesmer lab
