57] Procedures for expression, modification, and analysis of human fibroblast interferon (IFN-β) genes in heterologous cells

Abstract

Publisher Summary This chapter describes procedures used for expressing human interferon (IFN)-β in CHO cells, modifying the gene sequence encoding the carbohydrate attachment site of IFN-β, and analyzing the glycosylation state of the IFN-β produced. Human IFN-β, in contrast to multiple subspecies of IFN-α, is a glycoprotein with a single potential N-linked carbohydrate attachment signal located at residues 80–82. The physical properties and specific activity of E. coli IFN-β differ significantly from those of its natural counterpart. Indirect methods with tunicamycin or other metabolic inhibitors of glycosylation suggest that some biologically active, unglycosylated IFN-β is produced and secreted into the media in the presence of such inhibitors. Glycoproteins are known to bind to concanavalin A (con A) and can therefore be separated from nonglycosylated proteins by chromatography on Con A-sepharose. The 18,500 D form of IFN-β produced from the pMB-1 gene appears to be unglycosylated confirming that the asparagine residue at position 80 is the site for glycosylation in native IFN-β. Preliminary results indicate that the unglycosylated IFN-β produced and secreted from the mutant gene has a much lower specific biological activity than glycosylated IFN-β. The availability of these cell lines permits to evaluate further the role of glycosylation in the activity and physical properties of IFN-β.