title: Purification and characterization of recombinant human interleukin 5 expressed in Chinese hamster ovary cells.
abstract: Recombinant human interleukin 5 (rhIL-5) expressed in Chinese hamster ovary cells was purified and characterized. Molecular heterogeneity was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two major components of Mr around 40,000 were detected under non-reducing conditions. However, under reducing conditions, the Mr of rhIL-5 was determined to be 22,000 and 20,000. After treatment with endoglycosidase F, a band with an apparent Mr of 18,000 was observed. Treatment of rhIL-5 with 2-mercaptoethanol followed by N-ethylmaleimide resulted in its dissociation into a monomeric form. This alkylated rhIL-5 was biologically less active than intact rhIL-5. These results suggest that rhIL-5 exists as a dimer, and that the heterogeneity of rhIL-5 is mainly due to the difference in the content of carbohydrate. Moreover, the formation of disulfide bond(s) might be important for the biological activity of rhIL-5.
authors.affiliation: Suntory Institute for Biomedical Research, Osaka.
authors.forename: M
authors.initials: M
authors.lastname: Tsujimoto
authors.forename: H
authors.initials: H
authors.lastname: Adachi
authors.forename: S
authors.initials: S
authors.lastname: Kodama
authors.forename: N
authors.initials: N
authors.lastname: Tsuruoka
authors.forename: Y
authors.initials: Y
authors.lastname: Yamada
authors.forename: S
authors.initials: S
authors.lastname: Tanaka
authors.forename: S
authors.initials: S
authors.lastname: Mita
authors.forename: K
authors.initials: K
authors.lastname: Takatsu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D006224:Cricetinae; D004220:Disulfides / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006026:Glycoside Hydrolases / Q000378:metabolism; D015848:Interleukin-5; D007378:Interleukins / Q000032:analysis* / Q000302:isolation & purification; D007525:Isoelectric Focusing; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010053:Ovary / Q000166:cytology; D011994:Recombinant Proteins / Q000302:isolation & purification*
chemical_list: D004220:Disulfides; D015848:Interleukin-5; D007378:Interleukins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase