Glycoengineering tobacco plants to stably express recombinant human erythropoietin with different N-glycan profiles

Plant-based expression system has many potential advantages to produce biopharmaceuticals, but plants cannot be directly used to express human glycoproteins because of their differences in glycosylation abilities from mammals. To exploit plant-based expression system for producing recombinant human erythropoietin (rhuEPO), we glycoengineered tobacco plants by stably introducing seven to eight mammalian genes including a target human EPO into tobacco in order to generate capacities for β1,4-galactosylation, bisecting N-acetylglucosamine (GlcNAc) and sialylation. Wild type human β1,4-galactosyltransferase gene (GalT) or a chimeric GalT gene (ST/GalT) was co-expressed to produce rhuEPO bearing β1,4-galactose-extended N-glycan chains as well as compare their β1,4-galactosylation efficiencies. Five mammalian genes encoding enzymes/transporter for sialic acid biosynthesis, transport and transfer were co-expressed to build sialylation capacity in plants. The human MGAT3 was co-expressed to produce N-glycan chains with bisecting GlcNAc. Our results demonstrated that the above transgenes were incorporated into tobacco genome and transcribed. ST/GalT was found to be more efficient than GalT for β1,4-galactosylation. Furthermore, co-expressing MGAT3 generated N-glycans likely bearing bisected GlcNAc. However, our current efforts did not result in generating sialylation capacity. Created transgenic plants expressing EPO and ST/GalT could be used to produce rhuEPO with high proportion of β1,4-galactose-extended N-glycan chains for tissue protective purposes.