Dear Wenhe, >From my perspective you have quite a few options. Notably, the presence of glycosylation in the native host does not always (or even often) mean that you *must* have the protein in the native glyco-state in order to be 'right'. Very often glycosylation sites can be trimmed (by e.g. Kifunensine and similar compounds) or mutated out. When one mutates glycosylation sites one runs a significant risk of stopping export - meaning that without correct glycosylaton some proteins do not secrete. This is very much protein dependent and host dependent but I have personally experienced about a dozen cases (out of hundreds which went just fine). So it's not a #1 item to be scared of.
Options: 1. refold from IB in E. coli 2. secrete into periplasm in E. coli 3. secrete into media from a gram-positive organism (e.g. Bacillus) 4. secrete from yeast 5. secrete from insect cells 6. secrete from mammalian cells For you it seems that 5 and 6 may be a bit of an overkill, but it all depends on the nature of the protein you're working with :) I normally recommend 1 and 6 to 'bracket' the probability spread (1 is very easy but often not very successful whereas 6 is expensive but is often very successful, and both are very fast). Artem - Cosmic Cats approve of this message On Wed, Jul 11, 2018 at 11:54 PM, 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
