Brief comment based on single experiment with the CHO-Lec-: In principle the cells are compromised, so expect lower yields, but a single N-clyc site might be a good omen. In our hands with 5 sites on 70 kDa, the patterns had clearly reduced but still significant complexity per MS/HPLC glycan analysis. In any case, the remaining glycans were significantly more complex than what we expected from reading the literature - which might be an advantage in your case. In ours it was not :-/
Best, BR -----Original Message----- From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Bärbel Blaum Sent: Mittwoch, 15. Oktober 2014 15:57 To: [email protected] Subject: Re: [ccp4bb] Trimming of carbohydrate chains Depending on how you produce the protein you might want to try CHO-Lec cells which are only able to produce the high mannose core structures of N-glycans as they lack further transferases. Further treatment with Endo H leaves only the reducing end mannose of the N-glycan chains. Some other cleaving enzymes can be found at NEB https://www.neb.com/applications/glycobiology/removal-of-n-linked-and-o-linked-glycans-from-proteins or other companies, of course. It might be good to consult chapter 8 (and 9 for o-glycans) of the glycobiology "bible", available on the NCBI bookshelf http://www.ncbi.nlm.nih.gov/books/NBK1908/ to understand what each of these enzymes is doing. Good luck, Baerbel Quoting "A. Radu Aricescu" <[email protected]>: > Dear Herman, > > Assuming that the protein is eukaryotic, and glycans N-linked, various > ways of dealing with the issue are described in PMID: > 17355862. > > Best wishes, > > radu > > > ------------------------------------------ > A. Radu Aricescu, PhD > MRC Senior Research Fellow > Associate Professor > > University of Oxford > Wellcome Trust Centre for Human Genetics Division of Structural > Biology Roosevelt Drive, Oxford OX3 7BN United Kingdom > Phone: +44-1865-287564 > Fax: +44-1865-287547 > https://www.strubi.ox.ac.uk/research/a-radu-aricescu > > > ---- Original message ---- >> Date: Wed, 15 Oct 2014 13:03:13 +0000 >> From: CCP4 bulletin board <[email protected]> (on behalf of >> [email protected]) >> Subject: [ccp4bb] Trimming of carbohydrate chains >> To: [email protected] >> >> 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 > -- Bärbel Blaum, Ph.D. Interfakultäres Institut für Biochemie (IFIB) Hoppe-Seyler-Strasse 4 D-72076 Tübingen Germany +49 70 71 29 73 375
