C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
195/1.22, 195/1.
C12N 15/09 (2006.01) C07H 21/04 (2006.01) C07K 14/545 (2006.01) C12N 1/21 (2006.01) C12N 15/25 (2006.01) C12N 15/81 (2006.01) C12P 21/02 (2006.01)
Patent
CA 1340978
Interleukin 1 is purified to homogeneity by use of various techniques including ion exchange chromatography and dye-ligand affinity chromatography, thereby enabling the amino acid composition of this protein to be ascertained and its amino acid sequence to be partially determined. Double-stranded cDNA is prepared from polyadenylated RNA extracted from activated human peripheral blood adherent mononuclear cells. The cDNA is inserted within a plasmid vector and then the recombinant plasmid employed to transform an appropriate host. Plasmid DNA prepared from pools of transformed hosts is hybridized with a labeled, synthetic oligonucleotide probe corresponding to a portion of the ascertained amino acid sequence of the interleukin 1 protein. Pools of host cells that provide a positive signal to the probe are identified, plated out and then employed in direct bacterial colony hybridization with the same probe, thereby to isolate the particular positive colony. Plasmid DNA is prepared from this colony and characterized by restriction enzyme mapping and sequenced. The coding region for the IL-1 gene is inserted into a shuttle vector for amplification of the vector followed by expression of functional IL-1.
476035
Cerretti Douglas P.
Conlon Paul J. III
Cosman David J.
Grabstein Kenneth H.
Hopp Thomas P.
Cistron Biotechnology Inc.
Immunex Corporation
Smart & Biggar
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