title: Production and purification of recombinant human interleukin-5 from yeast and baculovirus expression systems.
abstract: A cDNA for human interleukin-5 (hIL-5) was created from the hIL-5 gene using site-directed mutagenesis to splice out the introns in vitro. This cDNA was expressed in yeast and baculovirus systems, utilizing in both cases an in-frame fusion to the pre sequence of the alpha-mating-type factor to direct secretion. The highest level of production was achieved from Sf9 cells using a baculovirus vector in serum-containing medium (2.7 mg/l), whereas in serum-free medium ten times less hIL-5 was produced. In the yeast system much lower levels of hIL-5 were produced (12.5 micrograms/l). Recombinant hIL-5 was purified to homogeneity from serum-free baculovirus cultures. The rhIL-5 consisted of a 30-kDa homodimer linked by disulfide bridging. The purified recombinant protein had a specific activity on murine BCL1 cells of 1.5 x 10(4) U/mg, of 3 x 10(5) U/mg in the murine eosinophil differentiation factor assay, and 2.4 x 10(7) U/mg in a human peripheral eosinophil maintenance assay.
authors.affiliation: Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra.
authors.forename: E
authors.initials: E
authors.lastname: Ingley
authors.forename: R L
authors.initials: RL
authors.lastname: Cutler
authors.forename: M C
authors.initials: MC
authors.lastname: Fung
authors.forename: C J
authors.initials: CJ
authors.lastname: Sanderson
authors.forename: I G
authors.initials: IG
authors.lastname: Young
mesh_terms: D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D006801:Humans; D015848:Interleukin-5 / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D015848:Interleukin-5; D011994:Recombinant Proteins