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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 hIgE-Fc bind to the high-affinity IgE receptor, Fc epsilon RI, with about the same affinity as myeloma IgE (Ka in the range 10(10)-10(11) M-1), and were able to sensitize isolated human basophils for anti-IgE triggering of histamine release. However, only the double mutant hIgE-Fc approached the affinity of myeloma IgE for the low-affinity receptor, Fc epsilon RII (Ka = 7.3 x 10(7) M-1), whereas the wild-type hIgE-Fc bound with a 10-fold lower affinity (Ka = 4.1 x 10(6) M-1).

Type

Journal article

Journal

Protein Eng

Publication Date

02/1995

Volume

8

Pages

193 - 199

Keywords

Amino Acid Sequence, Animals, Base Sequence, CHO Cells, Cricetinae, Deoxyribonuclease EcoRI, Deoxyribonucleases, Type II Site-Specific, Electrophoresis, Polyacrylamide Gel, Glycosylation, Histamine Release, Humans, Immunoglobulin E, Immunoglobulin Fc Fragments, Macromolecular Substances, Molecular Sequence Data, Mutagenesis, Site-Directed, Recombinant Proteins, Structure-Activity Relationship, Transfection