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

Journal article

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).

Authors: Young, R; Owens, R; Mackay, G; Chan, C; Shi, J

• Publication status: Published
• Journal: Protein engineering
• Volume: 8
• Issue: 2
• Pages: 193-199
• Publication date: 1995-02-01
• DOI: 10.1093/protein/8.2.193
• EISSN: 1460-213X
• ISSN: 0269-2139
• Language: English
• Keywords: Humans; Animals; Transfection; Amino Acid Sequence; Electrophoresis, Polyacrylamide Gel; Histamine Release; Glycosylation; CHO Cells; Structure-Activity Relationship; Recombinant Proteins; Mutagenesis, Site-Directed; Base Sequence; Molecular Sequence Data; Deoxyribonuclease EcoRI; Immunoglobulin Fc Fragments; Macromolecular Substances; Immunoglobulin E; Deoxyribonucleases, Type II Site-Specific; Cricetinae