Abstract: A chemically-synthesized oligonucleotide composing a portion of the nucleotide sequence of the human IL-2 is employed as a probe to isolate the gene coding for human IL-2. The human IL-2 gene is selected from a cDNA library prepared from RNA produced by mitogen-stimulated Jurkat cells. Double-stranded cDNA is prepared from polyadenylated RNA extracted from bovine cells thought to produce interleukin-2. Such cDNA is inserted within a plasmid vector and the recombinant plasmid employed to transform hosts. Plasmid DNA, prepared from pools of the transformed hosts, is hybridized with a probe composed of a large portion of the coding sequence of the human IL-2 gene. Pools of host cells that provide signal to the human cDNA probe are identified, subdivided, and rescreened until a single positive colony is identified. Bovine plasmid cDNA is prepared from this colony, and the bIL-2 gene is sequenced.
Type:
Grant
Filed:
July 31, 1986
Date of Patent:
November 21, 1989
Assignee:
Immunex Corporation
Inventors:
Dirk M. Anderson, Paul E. Baker, Michael A. Cantrell, Douglas P. Cerretti, David J. Cosman, Steven D. Gimpel, Kenneth H. Grabstein, Alf D. Larsen, Kate N. McKereghan
Abstract: Cloning and expression of nucleotide DNA segments encoding bovine IL-1.beta., and processes for producing purified bovine IL-1.beta. as a product of recombinant cell culture, are disclosed.
Type:
Grant
Filed:
March 13, 1987
Date of Patent:
November 7, 1989
Assignee:
Immunex Corporation
Inventors:
Douglas P. Cerretti, Brian S. Davis, Charles R. Maliszewski
Abstract: A process is disclosed for purifying a recombinant fusion protein having an N-terminal identification sequence comprising multiple anionic amino acid residues, comprising forming a complex of the protein with a divalent cation dependent monoclonal antibody specific for the sequence, isolating the complex, and dissociating antibody and protein by selectively depleting the concentration of divalent cations in contact with the complex. A particular calcium-dependent monoclonal antibody, 4E11, is useful in an embodiment of the process which employs the identification peptide DYKDDDDK.
Abstract: A process is provided for purifying recombinant IL-1 from microbial cells, comprising suspending the cells in an aqueous buffered medium having a pH from about 1 to about 5; disrupting the cells to provide an extract containing solubilized IL-1; and recovering solubilized Il-1 from the extract.