https://www.genovis.com/immobilized-sialexo-landing-page/?gclid=Cj0KCQjw09HzBRDrARIsAG60GP_1kTDFogTI3q-xdRzMt5-ttHHGb4H7VQALwVhc8-5esGpLfNaSIs4aAhEWEALw_wcB

You may know that only eukaryotic cells glycosylate their proteins. Bacteria like E. coli will never do it. So the first thing is that you need to make sure your expression system is eukaryotic.

Second, your glycoprotein should have naturally occurring N-glycosylation sites. These are specific motifs in your amino acid sequence. They contain the amino acid sequence "N X (S/T)", where N is the asparagine that becomes glycosylated, X is any amino acid and S/T is serine or threonine. You can use the website netNglyc to find them in your sequence.

Finally, glycosylation happens in the endoplasmic reticulum and golgi apparatus. That means your protein must be targeted there. That's accomplished with a "secretion signal" aka "leader peptide" in the front of your sequence. You can find it with another website, SingalP-5.0.

That's all you need. If you have those three things, a eukaryotic cell should glycosylate your protein.

Glycosylated protein expression in prokaryotes (https://patents.google.com/patent/US9512434B2/en)

Optimal and consistent protein glycosylation in mammalian cell culture (https://academic.oup.com/glycob/article/19/9/936/1988144)

John Schloendorn Roger Davis Suhad Mohammed

Hi everyone, thanks for your help - I am a lot less stuck now. Just one more question, i have got as far as putting my gene into the vector and finding a cell line to over-express my protein. How would i go about purifying my glycoprotein. For example if I had grown my DNA on agar plates - what would my next step be, or would it be better to grow the protein in suspension cells - either way how would I purify? Thanks