Expression of human interferon-alpha 2 in Sf9 cells. Characterization of O-linked glycosylation and protein heterogeneities.
Human interferon alpha 2 (IFN-alpha 2) was expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression system. The protein purified by immunoaffinity chromatography exhibited biological activity identical to that of leukocyte-derived 'natural' IFN-alpha 2. However, the protein was found to be heterogeneously glycosylated, partially truncated by proteolysis and partially lacking a disulfide bridge. The major product was shown to be O-glycosylated at the same position as natural human IFN-alpha 2. Enzymatic cleavage, reverse-phase HPLC peptide mapping and plasma-desorption mass spectroscopy analysis revealed the presence of two types of O-linked carbohydrates. The major O-linked carbohydrate was found to be the disaccharide galactosyl(beta 1-3)-N-acetylgalactosamine, the minor component the monosaccharide N-acetylgalactosamine. No evidence for sialylation was found. The non-glycosylated species representing about 40% of the total material were shown to partially lack the C-terminal three amino acids. In addition an unglycosylated, reduction-sensitive dimer was observed. This was formed due to the lack of the N-terminal cysteine normally forming an intramolecular disulfide bridge. Furthermore, a minor species was identified which contains Cys1 and Cys98 in a modified form, thereby hindering the formation of a disulfide bridge between these two residues.
Sugyiama
K
K
Ernst-Boehringer Institut für Arzneimittelforschung, Department of Protein Chemistry, Bender & Co., Vienna, Austria.
Ahorn
H
H
Maurer-Fogy
I
I
Voss
T
T
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths; D010449:Peptide Mapping; D011487:Protein Conformation
D002241:Carbohydrates; D017931:DNA Primers; D016898:Interferon-alpha