title: Characterization of E. coli-derived recombinant human interferon-beta as compared with fibroblast human interferon-beta.
abstract: Homogeneous E. coli-derived recombinant human interferon-beta (E. coli-rHuIFN-beta) was characterized in order to elucidate its physicochemical properties, as compared with those of fibroblast human interferon-beta (fibroblast HuIFN-beta). Purified E. coli-rHuIFN-beta and fibroblast HuIFN-beta exhibited a single band of Mr 19,000 and 23,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The primary structure of E. coli-rHuIFN-beta was identical to the prediction from the cDNA sequence. Furthermore, both the circular dichroism (CD) spectra and the 1H nuclear magnetic resonance (NMR) spectra of E. coli-rHuIFN-beta and fibroblast HuIFN-beta at pH 6.8 were closely similar to each other. On the other hand, on reverse-phase high-performance liquid chromatography (HPLC) using a C18 column, the retention time of E. coli-rHuIFN-beta was longer than that of fibroblast HuIFN-beta. Moreover, although the isoelectric point of E. coli-rHuIFN-beta was pH 8.9, purified fibroblast HuIFN-beta exhibited multiple isoelectric points, probably due to heterogeneity of the carbohydrate moiety. These results indicate that the E. coli-rHuIFN-beta polypeptide folds similarly to fibroblast HuIFN-beta, and the carbohydrate moiety of natural HuIFN-beta has little influence on higher-order structure but does influence the hydrophobic and the electrostatic properties of the molecule.
authors.affiliation: Basic Research Laboratories, Toray Industries, Inc. Kanagawa.
authors.forename: J
authors.initials: J
authors.lastname: Utsumi
authors.forename: S
authors.initials: S
authors.lastname: Yamazaki
authors.forename: K
authors.initials: K
authors.lastname: Hosoi
authors.forename: S
authors.initials: S
authors.lastname: Kimura
authors.forename: K
authors.initials: K
authors.lastname: Hanada
authors.forename: T
authors.initials: T
authors.lastname: Shimazu
authors.forename: H
authors.initials: H
authors.lastname: Shimizu
mesh_terms: D000595:Amino Acid Sequence; D004247:DNA / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007370:Interferon Type I / Q000235:genetics; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins
chemical_list: D007370:Interferon Type I; D011994:Recombinant Proteins; D004247:DNA