Engineering mammalian cells to produce plant-specific N-glycosylation on proteins

Abstract

Protein N-glycosylation is an essential and highly conserved posttranslational modification found in all eukaryotic cells. Yeast, plants and mammalian cells, however, produce N-glycans with distinct structural features. These species-specific features not only pose challenges in selecting host cells for production of recombinant therapeutics for human medical use but also provide opportunities to explore and utilize species-specific glycosylation in design of vaccines. Here, we used reverse cross-species engineering to stably introduce plant core α3fucose (α3Fuc) and β2xylose (β2Xyl) N-glycosylation epitopes in the mammalian Chinese hamster ovary (CHO) cell line. We used directed knockin of plant core fucosylation and xylosylation genes (AtFucTA/AtFucTB and AtXylT) and targeted knockout of endogenous genes for core fucosylation (fut8) and elongation (B4galt1), for establishing CHO cells with plant N-glycosylation capacities. The engineering was evaluated through coexpression of two human therapeutic N-glycoproteins, erythropoietin (EPO) and an immunoglobulin G (IgG) antibody. Full conversion to the plant-type α3Fuc/β2Xyl bi-antennary agalactosylated N-glycosylation (G0FX) was demonstrated for the IgG1 produced in CHO cells. These results demonstrate that N-glycosylation in mammalian cells is amenable for extensive cross-kingdom engineering and that engineered CHO cells may be used to produce glycoproteins with plant glycosylation.

Identification of Genomic Safe Harbors in the Anhydrobiotic Cell Line, Pv11

“…For example, in basic research, a variety of genetic materials have been integrated into a mouse GSH, the Rosa26 locus, allowing exogenous gene expression or highly targeted gene knockdown experiments [ 7 ]. In applied research, GSHs have been used to establish cells that can stably produce proteins of medical interest, for example, therapeutic antibodies [ 4 , 8 ] and species-specific glycosylated proteins [ 9 ]. In addition, some researchers have proposed a therapy for genetic diseases using cells that are genetically modified via human GSHs [ 10 ].…”