Immunex Corporation

Other

Steven D Gimpel

Dirk M Anderson

Douglas Pat Cerretti

David J Cosman

Kenneth H Grabstein

Alf D Larsen

Paul E Baker

Kate N McKereghan

Michael A Cantrell

Virginia L Price

Michael C Deeley

Amplified expression of recombinant DNA products is achieved in hosts expressing protease that cleave at multi-basic amino acid residues. To this end, wild-type genes encoding the desired protein products are mutated by substituting codons or eliminating codons encoding multi-basic amino acid residues while maintaining the activity of the expressed protein product. Mutation of the desired gene can be conveniently carried out by site-specific in vitro mutagenisis.

Analogs of human granulocyte-macrophage colony stimulating factor

A process for preparing bovine interleukin-2 (bIL-2) by culturing a microbial host transformed by a vector containing a DNA sequence encoding bIL-2 under conditions suitable for the expression of bIL-2 and recovering bIL-2.

Process for preparing bovine interleukin-2

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. The plasmid DNA is employed to express recombinant bovine IL-2 in yeast and bacterial expression systems, with the expressed bovine IL-2 purified to homogeneity by one or more reverse phase high-performance liquid chromatography procedures.

DNA sequences encoding bovine interleukin-2

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Seattle, WA, United States of America

Steven D Gimpel