title: Production and purification of recombinant human interleukin-6 secreted by the yeast Saccharomyces cerevisiae.
abstract: The coding region of the human interleukin-6 (hIL6) gene was fused to the prepro secretion signal of the alpha-mating factor gene in several yeast host strains. It was found that the KEX-2 protease was unable to cleave the prepro-Lys-Arg-Pro-IL6 sequence, but that unspecific cleavage of the precursor protein had occurred. The prepro-Lys-Arg-Ala-Pro-IL6 sequence, however, was correctly recognized and cleaved by the KEX-2 protease, and IL6 was efficiently secreted into the culture medium. The N-terminal Ala-Pro peptide was removed during processing by wild-type yeast strains, but was retained in a ste13 mutant. IL6 as well as the aberrant proteins were not glycosylated. The transformed cells could secrete up to 30 micrograms/ml IL6. The protein was purified from the medium to homogeneity by ion-exchange chromatography and gel filtration, and had a specific activity of about 2 x 10(8) IU/mg in a proliferation assay.
authors.affiliation: Laboratory of Molecular Biology, State University of Gent, Belgium.
authors.forename: Y
authors.initials: Y
authors.lastname: Guisez
authors.forename: B
authors.initials: B
authors.lastname: Tison
authors.forename: J
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: J
authors.initials: J
authors.lastname: Demolder
authors.forename: G
authors.initials: G
authors.lastname: Bauw
authors.forename: G
authors.initials: G
authors.lastname: Haegeman
authors.forename: W
authors.initials: W
authors.lastname: Fiers
authors.forename: R
authors.initials: R
authors.lastname: Contreras
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002853:Chromatography, Liquid; D004271:DNA, Fungal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000032:analysis; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D000596:Amino Acids; D004271:DNA, Fungal; D004731:Endotoxins; D015850:Interleukin-6; D011994:Recombinant Proteins