Question

Imagine you have discovered that a specific growth factor shows antitumoral properties. To study these properties in more detail, you need to synthetize large quantities of it and you use recombinant DNA technology in E. coli to do so. However, your recombinant protein does not have the same properties than the endogenous one. You confirm that the protein sequences are an exact match. Do you think that there could be glycosylation differences between the two proteins? Would you suspect their differences could be due to glycosylation if it would be a cytoplasmic factor? Think on an experiment to 1) ensure that the recombinant protein has the same glycosylation than the endogenous protein, and 2) modify the endogenous protein to test if its function depends on its glycosylation

Answer 1

Yes there will be difference in glycosylation patterns in bacteria and eukaryotes

Bacteria lacks oligosaccharide transferase mediated N glycosylation and it performs cytoplasmic stepwise glycosylation whereas eukaryote has oligosaccharide transferase mediated N glycosylation

Eukaryote lacks oligosaccharide transferase mediated O glycosylation and eukaryote lacks it and it performs cytoplasmic O glycosylation

2. Yes there could be difference since bacteria does not perform cytoplasmic O glycosylation

3. 1) Instead of bacteria, we can synthesize the protein in eukarytic organism such as yeast. Express the recombinant protein in yeast or other eukaryotes and observe the glycosylation pattern

2) prepare 1 control endogenous protein and in sample 1 remove glycosylation sites in the protein and sample 2 modify the glycosylation pattern as that of bacteria and observe for difference between control and samples. If function not changed, we can use bacteria itself