HIGH-LEVEL EXPRESSION AND PURIFICATION OF A RECOMBINANT HUMAN ALPHA-1,3-FUCOSYL-TRANSFERASE IN BACULOVIRUS-INFECTED INSECT CELLS

A human alpha-1,3-fucosyltransferase (Fuc-TVII) was expressed by recom binant baculovirus-infected insect Sf9 cells as a secretory fusion pro tein. The fusion protein consisted of the human granulocyte colony-sti mulating factor signal peptide followed by an IgG-binding domain of pr otein A, a Fuc-TVI-derived peptide, and the putative catalytic domain of Fuc-TVII, The signal peptide was correctly cleaved and the recombin ant Fuc-TVII was secreted into the culture medium at a concentration o f 10 mu g/ml. The recombinant Fuc-TVII could be highly purified in a s ingle-step purification procedure, i.e., IgG-Sepharose column chromato graphy. The enzymatic properties of the Sf9-produced Fuc-TVII were com pared with the properties of that expressed by a human B-cell line, Na malwa KJM-1, transfected with an episomal plasmid carrying the fusion Fuc-TVII cDNA. Both recombinant proteins showed alpha-1,3-fucosyltrans ferase activity toward a type II oligosaccharide with a terminal alpha -2,3-linked sialic acid among various accepters. The apparent K-m valu es of Sf9-produced Fuc-TVII for GDP-fucose and its acceptor substrate were slightly lower than those of the Fuc-TVII produced by Namalwa KJM -1 cells. Sf9-produced Fuc-TVII has N-linked carbohydrate chains whose molecular weights are lower than those linked to Namalwa KJM-1-produc ed Fuc-TVII. This difference in carbohydrate structure hardly affects the thermal stability of Fuc-TVII. The baculovirus expression system i s available for high-level expression of stable and enzymatically acti ve secretory Fuc-TVII, (C) 1997 Academic Press.