I'm a student and new to the forum and wondered if anyone could give me some advice.
I have a glycoprotein fragment which I cloned into a retroviral expression vector. This has expressed well and the proteins fine for binding studies etc. The problem I have is that the fragment is N glycosylated (only 1 site) and when it runs on a gel it runs as a tight doublet. The top band is the glycosylated protein.
In the long-term I hoped to maybe set up some crystal trials using the protein as it expresses so well but I've been told glycosylation is a big no no.
Looking at the literature, groups who have crystalised the protein have used either baculoviral or bacterial vectors which I assume was partly to get over the glycosylation. The fragment I have was a bit of a nightmare to clone so I'm reluctant to go back and re-design primers. I have it in the Topo 2.1 TA vector and the fragment has:
AgeI and PacI site
XhoI and BamHI site
We have the pET 28a and pET 48b vectors in the lab. On the multiple cloning sites there is a BamHI and XhoI site so I thought maybe I could double digest my fragment out of Topo and then ligate into double digested pET vector for expression in E-coli?
My XhoI and BamHI sites are back to front and hope this question doesn't sound too silly but, does it matter which way round my fragment goes into the pET vector?
Which pET vector would be most recommended?
Are there any other bacterial expression vectors which may be complementary to my restriction sites?
Any advice would be much appreciated!