Dear Bulletin Board, as usual with the bulletin board, I quickly got a number of very useful suggestions, which I summarize below.
Mick Blaise, Alisa Glukhova and Jacinto suggested to use endo F. Mick said that in his case always some sugar was left, which in my case would keep my protein in solution. Jacento also suggested endo F3, which would be less aggressive and leave one NAG attached to the protein. Alisa warned that endo F could still cleave sugars while boiling the samples for SDS-PAGE, so one should watch out while working out the cleaving conditions. Baerbel Blaum and Roberto Steiner suggested endo H, which would leave only the reducing end mannose of the N-glycan chains. Baerbel and Alisa also suggested CHO-lec cells, which have glycosylation defects and produce less glycosylated protein. Here yields may be lower and the remaining carbohydrate may still be more complex as expected (Bernard Rupp). Brad Graves and Jacinto suggested mutating away carbohydrate sites. Brad had looked at the same protein from other species to find out whether the sites were conserved. It turned out that the non-conserved sites could be mutated away without problems. Thanks to everybody who reacted! Herman -----Ursprüngliche Nachricht----- Von: Mickael Blaise [mailto:[email protected]] Gesendet: Mittwoch, 15. Oktober 2014 15:16 An: Schreuder, Herman R&D/DE Betreff: Re: [ccp4bb] Trimming of carbohydrate chains Dear Herman we usually trim the sugars after purification (from baculovirus expression) using PnGase F with a one to one ratio in mass (we purify ourself the PNGase F so it is cheap) with an O.N. incubation at room temp as far I can see form the crystal structures solved after triming the sugars, there is always some GlcNAc or GlcNAc-GlcNAc left in our case triming more is making the protein instable but not triming at all does not yield to good crystal hope it helps regards Mick Mickael Blaise PhD CARB Centre Dept. of Molecular Biology and Genetics Aarhus University Gustav Wieds Vej 10c DK-8000 Aarhus C - DENMARK Phone: + 45 8715 5524 On Oct 15, 2014, at 3:03 PM, [email protected] 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
