First, I don't think glycosylation occurs on lysine residues. You should try to crystallize it as is, if that doesn't work, try to remove the glycosylation with some or all of the following: EndoH, PNGaseF, EndoF1, EndoF3. We have used them all to various degrees of efficiency to remove glycans from insect-expressed proteins. It may or may not crash out, that is protein dependent. You can also mutagenize the potential N-linked sites which has been very successful for me.
There is no way to say if an insect signal sequence works better then the native signal sequence, the only way to know is to try both. If you have a lot of protein in the cells, that might suggest signal sequence cleavage is inefficient, and perhaps and insect signal sequence would be better. Sometimes adding a gly-gly linker after the signal sequence will improve cleavage. If you do deglycosylate I wouldn't worry about purifying the deglycosylated from glycosylated, just let the crystallization experiment select for the crystallizable material. In other words, separating different glycoforms is like any other purification, you just have to keep trying things until you get the results you want. In theory a lectin column would do it, but those aren't 'high-resolution' separations (in my hands...) In short, I would try everything you can think of until you get good crystals. Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Fri, Apr 8, 2011 at 2:06 PM, joybeiyang <joybeiy...@gmail.com> wrote: > > Dear All, > > I am currently working with a protein which is glycosylated on several N and > K. I use insect cells for secretion expression, so the protein was supposed > to be correctly glycosylated. However after several steps of purification, > there are still a minor doublet under the major band, which later on was > proved to be my target protein without glycosylation. My question is: > > 1. Should I neglect the doublet and just crystallize at this point? (The > unglycosylated form compare to the glycosylated form were about 5%:95%) > 2. Should I deglycosylate the protein before crystallization and if so could > anyone share with me a protocol? (Is precipitation likely to occur during > deglycosylation?) > 3. Was the unglycosylated form arise from cell death during cell culture or > was it arise from the overexpression of target protein and inadequacy of > glycosylation machinery and false secretion? How could I prevent it? Will a > insect specific signal peptide work better than the native signal peptide? > 4. Is it possible to separate the glycosylated and unglycosylated form > through either a monoQ/S or a Lectin Sepharose 4B column? > > Thank you very much for your input in advance and I greatly appreciate it! > > With all my best wishes, > > Bei > > 2011-04-08 > ________________________________ > joybeiyang
