Immunex Corporation

Cosman

Douglas Pat Cerretti

Carl J March

Steven K Dower

David L Urdal

Alf D Larsen

Interleukin-2 receptor derived from normal and malignant cells has been purified by use of various techniques including affinity chromatography in conjunction with a monoclonal antibody directed to the receptor. The purification process also includes reversed phased high performance liquid chromatography. By these techniques, interleukin-2 receptor has been purified to homogeneity. The high purification of the interleukin-2 receptor has made possible the sequencing of the amino acid residues at the N-terminal of this protein molecule. Double-stranded cDNA is prepared from polyadenylated RNA extracted from cell lines or other sources known to produce IL-2 receptor. The cDNA is inserted within a plasmid vector and then the recombinant plasmid employed to transform an appropriate host. Transformed hosts are identified and grouped into pools. Plasmid DNA prepared from these pools is hybridized with a labeled synthetic oligonucleotide probe corresponding to a portion of the amino acid sequence of the purified IL-2 receptor. The cDNA clone isolated with the probe is characterized by restriction enzyme mapping and sequenced by chain-termination method. The particular DNA clone that actually contains the gene coding for the functional IL-2 receptor is identified by expressing the clones in COS-7 monkey kidney cells and assaying for the expressed IL-2 receptor by its ability to bind IL-2 or a monoclonal antibody directed against the IL-2 receptor.

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Cosman