Recombinant protein antigens can be produced in a variety of different host systems (eg. E. coli, yeast, insect cells, mammalian cells). Microorganism hosts such as E.coli are generally the most successful, cost effective and in many cases the source of production may not significantly impact the success of the resulting antibodies. However, in cases where the antibody will be used to detect native epitopes such as in flow cytometry and the target protein contains post-translational modifications and/or has difficulty folding problems can arise.
We have illustrated these issues with the cell surface protein EGFR. EGFR is a multidomain protein with extensive disulfide bonds and glycosylation. We generated protein antigen for a single domain in both E. coli and mammalian cells and used them to immunize animals. The resulting antibodies were evaluated by flow cytometry on A431 cells (known to express high levels of EGFR).
While both hosts successfully produced the EGFR protein domain and generated an immune response to the antigen, a striking difference was seen in the flow cytometry analysis. Only the protein made in mammalian cells generated antibodies that efficiently detected the native EGFR in flow cytometry.