Purification and characterization of recombinant human IgE Fc epsilon fragment produced in mouse L cells.
A human IgE Fc epsilon fragment was isolated from the supernatant of the culture fluid of a recombinant mouse L cell line, L-IS11IgE-9. The purification procedure consisted of salting out from the supernatant with ammonium sulfate, immunoaffinity chromatography on a monoclonal antibody (E235I63)-Affi Gel 10 column, and gel filtration chromatography on a Sephacryl S-200 column. The final preparation represented a 5825-fold purification from the original culture fluid with a 25% recovery and about 3.1 mg of Fc epsilon fragment was obtained from 201 of culture fluid. The sp. act. of the purified preparation measured by the use of commercial human IgE determination kits was 0.93 x 10(6) units/mg protein. The purified preparation was homogeneous as judged by the end group analyses. The amino acid composition of the preparation coincided with that deduced from DNA sequence. The mol. wt of our preparation was about 110,000 under non-reducing conditions and 55,000 under reducing conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results showed that our preparation was a dimeric form having high reactivity against anti-human IgE antibodies.
Ikeyama
S
S
Biotechnology Laboratories, Takeda Chemical Industries Ltd, Osaka, Japan.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007073:Immunoglobulin E / Q000302:isolation & purification*; D007141:Immunoglobulin Fc Fragments / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification
D000596:Amino Acids; D007141:Immunoglobulin Fc Fragments; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D000645:Ammonium Sulfate