The protein shown below is a 173 kD, monomeric, glycosylated human membrane protein. I previously thought the best expression host to use was insect cells however given that insect cells expression hosts give different characteristic N-linked glycan structures of proteins compared to the typical mammalian proteins, would mamalian cells be the better choice? If the project goal was to raise antibodies against the protein after production, how could you simplify the expression approach?
Hello,
If you want to raise antybodies against the glycosylated parts of your protein that yes mamalian cells would probably be the best choice. If you just want antibodies against the protein no matter what part then if bacterias are able to produce your protein it would be a cheapest and easiest way to produce it. It is a membrane protein though but some strains are known to better produce membrane or toxic proteins (E coli C41 or C43). then There is a large panel of E coli strains that enables today the production of heterologous proteins in bacteria. Also cell free systems can be considered if you can have this possibility. Else if you already produce in insect cells can't it be possible to deglycosylate the protein? I think you can find some commercial mix for that.
Hope those ideas can help you. It really depends on what you exactly want with your protein and antybody and what you will do next with all this.
If you raise antibodies against the unglycosylated version it is possible that they will not work as their epitopes are masked by the glycosylation in vivo. Anyway, I would start to test expression in E. coli using only the extracellular domain, as this should be straight forward and leading to comparable high yields. Afterwards test whether these ABs work using a properly glycosylated protein, e.g. produced in a human cell line. You don't need large amounts of glycosylated protein in this case (a few µg for an ELISA maybe?) so this should be rather straight forward as well.
First, you need to prioritize your further steps. If this protein after purification needs to be tested in some functional bioassay, then you need to try to stick to the expression system which can produce oligosaccharides as close as possible to the native ones, because the structure of the oligosaccharides might influence the bioactivity.
Second, If the production of the specific antibodies is the primary goal, then you need to think about if the produced Abs would bind the natural antigen. Some Abs have very different specificities to the same protein depending on it's glycosylation extent.
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