Expression of human and murine interleukin-5 in eukaryotic systems.
A cDNA coding for murine interleukin-5 (IL-5) was isolated from the EL4.ExC5 cell line. With the exception of a single amino acid substitution at position 79 (Arg----His), it is identical to a published sequence. The coding sequence for human IL-5 was synthesized chemically, allowing the introduction of strategically located restriction enzyme cleavage sites. Both cDNAs were expressed in various eukaryotic systems. Deletion of the 3' untranslated region of the murine IL-5 gene led to a 5- to 10-fold increase in expression in Xenopus laevis oocytes and in NIH-3T3 cells. The highest production, however, was obtained in Sf9 cells using a baculovirus vector. Human IL-5 was obtained from transformed Saccharomyces cerevisiae as a secreted, mature form using an in-frame fusion to the leader sequence of alpha-mating type factor, and was purified to homogeneity. In all cases mentioned, IL-5 was found to be glycosylated, and its biological activity was dependent on a 40- to 50-kD homodimer configuration, linked together by disulfide bridges. Deglycosylation did not affect the biological activity. Recombinant human IL-5 is biologically active on some human B-CLL cells (proliferation in the presence of IL-2) and on murine BCl1 cells (proliferation) at a low specific activity (about 1-2 x 10(3) U/mg) and on human eosinophils (eosinophil peroxidase assay) at a high specific activity (at least 5 x 10(6) U/mg). Recombinant murine IL-5 from Sf9 cells has a specific activity of 1-2 x 10(7) U/mg in the BCl1 proliferation assay. An additive effect is seen in the presence of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and a synergistic effect in the presence of murine IL-4.
Tavernier
J
J
Roche Research Gent, Ghent, Belgium.
Devos
R
R
Van der Heyden
J
J
Hauquier
G
G
Bauden
R
R
Fache
I
I
Kawashima
E
E
Vandekerckhove
J
J
Contreras
R
R
Fiers
W
W
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005796:Genes; D006801:Humans; D015848:Interleukin-5; D007378:Interleukins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013045:Species Specificity; D014982:Xenopus laevis
D015848:Interleukin-5; D007378:Interleukins; D004247:DNA