Purification and characterization of natural human interleukin-3.
The human interleukin-3 gene was cloned in 1986 and the biochemical and biological properties of recombinant human interleukin-3 (rhuIL-3) protein were described. In this report we compare rhuIL-3 with nonrecombinant, natural huIL-3, purified from the supernatant of the human T cell leukemia line Jurkat. The main purification step, affinity chromatography, using a selected monoclonal anti-huIL-3 antibody, resulted in an approximately 40,000-fold enrichment of huIL-3. Combination of this step with ion-exchange and reverse-phase chromatography yielded natural huIL-3 of high purity (greater than 98%). A highly sensitive and specific sandwich ELISA, comprising two epitope-mapped monoclonal anti-huIL-3 antibodies, was used to quantitate huIL-3 during purification. Amino acid sequence determination revealed that the 38 N-terminal amino acids of Jurkat-derived huIL-3 are identical to the published sequence deduced from human fetal liver genomic DNA but differ in one residue from that derived from human T cell clones. The degree of glycosylation of Jurkat-derived huIL-3 was similar to Chinese hamster ovary cell-expressed rhuIL-3. Natural huIL-3 showed very similar biological activities to rhuIL-3 in proliferation and receptor binding assays utilizing huIL-3 responsive primary cells and cell lines, as well as in the human bone marrow colony assay.
Department of Preclinical Research, Sandoz Pharma Ltd., Basle, Switzerland.
G
G
Zenke
N A
NA
Lokker
U
U
Strittmatter
B
B
Fagg
S
S
Geisse
G
G
Huber-Wegmann
H P
HP
Kocher
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001681:Biological Assay; D002846:Chromatography, Affinity; D006031:Glycosylation; D006801:Humans; D007377:Interleukin-3 / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D007377:Interleukin-3; D011994:Recombinant Proteins