You may also want to have a look at this summary of a 2006 discussion: http://www.mail-archive.com/ccp4bb@dl.ac.uk/msg01697.html
From: joybeiyang Sent: Friday, April 08, 2011 2:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic: Is deglycosylation necessary for crystallization? 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
