Creating a triple mutant tobacco chassis with altered cfG expression for the production of humanized therapeutic protein

Plant-based expression systems offer a promising platform for producing therapeutic glycoproteins with human-compatible glycosylation patterns. This study aimed to engineer tobacco plants (Nicotiana tabacum cv. Yunyan 87) to modify glycosylation pathways for the production of glycoproteins with reduced immunogenicity, enhancing their potential for therapeutic applications. To achieve this, a 1257 bp fragment of the human β-1,4-galactosyltransferase (GALT) gene was cloned into the pHB vector and introduced into tobacco via Agrobacterium-mediated transformation. Four GALT-OE lines (13^#, 18^#, 22^# and 30^#) were generated which showed significantly higher GALT expression, especially GALT-OE 30^# which showed a 4.5-fold increase over wild-type (WT). Moreover, Western-blot and ELISA analyses showed that protein expression in galt13^#, and galt30^# was also increased. Triple mutants were generated by crossing the GALT-OE 30^# line with previously developed double mutants β-1,2-xylosyltransferase (CXT1P-RNAi) and α-1,3-fucosyltransferase (FUT4-RNAi), which showed a 70 % and 80 % reduction in CXT1P and FUT4 expression levels, respectively. The generated triple mutants (cfG028, cfG031, and cfG039) showed a 3.8-fold increase in GALT expression, and corresponding glycoprotein modifications at the protein level. This study establishes a foundation for the large-scale production of low-immunogenic recombinant glycoproteins with enhanced therapeutic efficacy using a tobacco-based system.