Characterization of four different mammalian-cell-derived recombinant human interferon-beta 1s. Identical polypeptides and non-identical carbohydrate moieties compared to natural ones.
In order to evaluate the structural identification of various recombinant human interferon-beta 1s, the recombinant proteins were produced in four different mammalian cells (human PC12 and PC8 lung adenocarcinoma cells, Chinese hamster ovary cells and mouse C127 cells) and characterized. Each mammalian-cell-derived recombinant human interferon-beta 1 represented a single band of 23 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, the same molecular mass as fibroblast-derived natural human interferon-beta 1. Specific activities, amino acid compositions, amino-terminal sequences, peptide maps on C18 reversed-phase high-performance liquid chromatography and circular dichroic spectra of recombinant proteins were in good agreement with natural ones. On the other hand, the patterns of isoelectric focusing were different between mammalian-cell-derived recombinant human interferon-beta 1s and natural human interferon-beta 1. Sugar composition analysis revealed that the recombinant protein from Chinese hamster ovary cells has a similar sugar composition to that of natural protein and the other recombinant proteins have increased amounts of galactose and glucosamine in comparison to the natural protein. Furthermore, there is no galactosamine in the natural protein, while small amounts of galactosamine were detected in the oligosaccharides released from PC8- and C127-derived recombinant proteins by N-glycanase. These results indicate that mammalian-cell-derived recombinant human interferon-beta 1s have identical polypeptides to those of natural human interferon-beta 1 but their carbohydrate moieties, including unusual N-linked oligosaccharides, are individually different from natural ones and depend on the host cell.
Utsumi
J
J
Basic Research Laboratories, Toray Industries, Inc., Kanagawa, Japan.
Mizuno
Y
Y
Hosoi
K
K
Okano
K
K
Sawada
R
R
Kajitani
M
M
Sakai
I
I
Naruto
M
M
Shimizu
H
H
D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis* / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006801:Humans; D007370:Interferon Type I / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D007525:Isoelectric Focusing; D008175:Lung Neoplasms; D008969:Molecular Sequence Data; D010053:Ovary; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics
D000596:Amino Acids; D002241:Carbohydrates; D007370:Interferon Type I; D010455:Peptides; D011994:Recombinant Proteins