Interferon-γ Receptor Extracellular Domain—IgG Fusion Protein Produced in Chinese Hamster Ovary Cells as Mixture of Glycoforms

Abstract

Glycosylation of proteins fulfills important functions and because of its diversity contributes to apparent protein heterogeneity. We investigated the heterogeneity of a fusion protein comprising the extracellular domain of the human interferon-γ (IFN-γ) receptor and parts of the human IgG₃ constant region, a potential IFN-γ antagonist. The protein was produced in Chinese hamster ovary (CHO) cells and was secreted into the culture medium as an 175 kD glycoprotein. Glycosylation represented approximately one-third of the apparent molecular mass of the fusion protein, consisted of N- and O-linked carbohydrate moieties, and included sialic acid residues as part of both N- and O-linked oligosaccharides. Fusion protein forms with different apparent molecular masses and charges were separated by ion-exchange chromatography. Preparative electrofocusing revealed a wide spectrum of glycoforms. Glycosylation of the fusion protein and of soluble IFN-γ receptors, comprising the extracellular domain of the native sequence, expressed in insect and CHO cells did not interfere with affinity of ligand binding.