I would not discard the insect expression right away. In yeast or similar you risk hyperglycosilation while sf tend to have paucimannose and relatively homogeneous glcnac2man5 decorations. It can be hard (and expensive) to homogeneously deglyc enzymatically but not sure if you also want to crystallize the prot. You can prep eg pngasef yourself.
Best br On Thu, Jul 12, 2018, 05:56 WENHE ZHONG <[email protected]> wrote: > Dear Community, > > A little bit out of topic here. An enzyme we like to use in our assay is > commercially available. However, we found some unfavorable activities that > probably from the contaminants from this commercial source. We checked the > company website and found out this enzyme was purified from the original > organism without tag for enhancing purification. No purity data was shown > as well. > > So we want to see whether we can produce the recombinant protein with tag. > After checking the literatures, this enzyme is a N-linked glycosylated > protein (glycan attached to 3 asparagine residues) and is from > fungi Penicillium citrinum. These 3 glycosylations probably affect protein > folding, stability and its location in the cell. Currently we have three > options here: > > 1. Express this enzyme in yeast which is a similar system compared to > Penicillium citrinum. His6 tag will be added to the N- or C- terminal of > this protein. > 2. Express this enzyme in the periplasm of E.coli. However, no > glycosylation system in E.coli. Only one paper published in a not well > known journal used this method. But the result is not very conclusive to > us. If their claim is correct, the glycosylation at this enzyme is not > essential for protein production. > 3. Express this enzyme in insect cells. More complicated and advanced > glycosylation system in insect cells compared to fungi. > > Anyone has experience with glycoprotein? It will be very helpful if I can > have your advice. Thank you. > > Kind regards, > Wenhe > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
