Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants

Abstract

We have constructed an expression vector that leads to secretion of the whole Fc of human immunoglobulin E (hIgE-Fc) from mammalian cells at levels up to 100 mg/l of culture. Two surface glycosylation sites at Asn265 and Asn371 have been changed to glutamine, to obtain a more homogeneous preparation of hIgE-Fc for structural studies. Comparison of wild-type and mutant products revealed that Asn371 is rarely glycosylated in Chinese hamster ovary cells. Both the double mutant and wild-type hIgEFc bind to the high-affinity IgE receptor, FcεRI, with about the same affinity as myeloma IgE (Ka in the range 1010-1011 M-1), and were able to sensitize isolated human basophils for anti-IgE triggering ..