title: Isolation and characterization of multiple variants of recombinant human interleukin 4 expressed in mammalian cells.
abstract: We have purified recombinant human interleukin 4 (huIL-4), formerly named B-cell stimulatory factor-1, from supernatants of COS-7 monkey kidney and L-929 cells transfected with the cDNA for huIL-4. The purified protein exhibited a specific activity of 2.6 X 10(7) units/mg in a T-cell proliferation assay and consisted of multiple components on sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibiting Mr values of 15,000, 18,000, and 19,000. All forms of huIL-4 eluted on gel filtration chromatography with an apparent Mr of 22,000. Gas-phase microsequencing identified 26 and 8 amino acid residues at the N and C termini, respectively, all of which were consistent with the cDNA sequence. The site of processing of the signal sequence was found to occur between Gly-24 and His-25. Incubation with N-glycanase converted the 18- and 19-kDa variants to a 15-kDa form. Treatment with endo-beta-N-acetylglucosaminidase H reduced the molecular mass of the 18-kDa variant to 15 kDa, but did not have any apparent effects on the mass of the 19-kDa species. The removal of oligosaccharide by any of these treatments did not affect bioactivity in the T-cell proliferation assay. Neither O-glycanase nor endo-beta-N-acetylglucosaminidase D affected the molecular weight of any of these species. These data suggest that differences in carbohydrate structure account, at least in part, for the observed microheterogeneity.
authors.affiliation: Department of Biotechnology, Schering Corporation, Bloomfield, New Jersey 07003.
authors.forename: H V
authors.initials: HV
authors.lastname: Le
authors.forename: L
authors.initials: L
authors.lastname: Ramanathan
authors.forename: J E
authors.initials: JE
authors.lastname: Labdon
authors.forename: C A
authors.initials: CA
authors.lastname: Mays-Ichinco
authors.forename: R
authors.initials: R
authors.lastname: Syto
authors.forename: N
authors.initials: N
authors.lastname: Arai
authors.forename: P
authors.initials: P
authors.lastname: Hoy
authors.forename: Y
authors.initials: Y
authors.lastname: Takebe
authors.forename: T L
authors.initials: TL
authors.lastname: Nagabhushan
authors.forename: P P
authors.initials: PP
authors.lastname: Trotta
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D006801:Humans; D015847:Interleukin-4; D007378:Interleukins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification*; D014162:Transfection
chemical_list: D007378:Interleukins; D011994:Recombinant Proteins; D015847:Interleukin-4; D004247:DNA