Effect of the developmental stage and tissue position on the expression and glycosylation of recombinant glycoprotein GA733-FcK in transgenic plants

Abstract

The influence of developmental stage and position (top, middle, and base) of leaves and stem tissues on the expression and glycosylation pattern of a recombinant therapeutic protein -GA733-FcK- was observed in transgenic seedlings during a 16-week growth period. RNA expression gradually increased with age in the middle and basal leaves and decreased in top leaves after 14 weeks. The protein expression level at all leaf positions increased until 14 weeks and slightly decreased at 16 weeks; it was lower in yellow leaves than in green leaves. In stem, protein expression gradually decreased from the top to the base. The glycosylation patterns of GA733-FcK were analyzed from 10 to 16 weeks. The plant-specific glycans increased in the top leaves at 14 weeks, but only slightly changed in the middle and basal leaves. The structure of glycans varied with tissue position. The glycosylation level in the top and middle leaves increased until 12 and 14 weeks, respectively, and decreased thereafter, whereas it decreased in basal leaves until 14 weeks and increased at 16 weeks. In stem, all three sections showed high-mannose type glycan structures. The area size of the glycans was significantly higher in the top stem than in both the middle and basal stems, and it was smaller in yellow leaves than in green leaves. The glycan profiles were similar between green and yellow leaves until 16 weeks. Thus, biomass-harvesting time should be optimized to obtain recombinant therapeutic proteins with ideal glycan structure profiles.

Murine response studies of insect cell (Sf9) expressed recombinant colorectal cancer vaccine candidate using surface plasmon resonance studies

“…; Lim et al . ). Oligomannose can enhance an immune response because of the interaction between oligomannose of glycoproteins and the mannose receptor on macrophages and dendritic cells.…”

“…These results suggest that the ER retention signal KDEL retains GA733-Fc in the ER without secretion to CM (in the absence of the signal peptide, the default secretion pathway was not followed). Attachment of KDEL has been used to enhance production of recombinant proteins with oligomannose glycan structure in animal, plant and insect cells (Henderson et al 1996;Schouten et al 1996;Lee et al 2013;So et al 2013;Kim et al 2015;Lim et al 2015). Oligomannose can enhance an immune response because of the interaction between oligomannose of glycoproteins and the mannose receptor on macrophages and dendritic cells.…”

Baculovirus titration method based on MOI values for optimizing recombinant protein expression of the anti‐cancer vaccine candidate GA733‐Fc using Sf9 insect cells

“…BEVS has many advantages, including scalability using high-density suspension cultures, capacity for expressing multiple genes at high expression levels, and the possibility of post-translational modifications (Deparis et al 2003;Malde & Hunt 2004;Kost et al, 2005). In the present study, we used the BEVS technology to express diverse recombinant proteins, including anti-colorectal cancer monoclonal antibody CO17-1A (Song et al 2010;Park et al 2011Park et al , 2014Lee & Ko 2014 and anti-cancer vaccine candidate GA733-Fc (Kim et al 2015;Park et al 2015;Qiao et al 2015;Lim et al 2015aLim et al , 2015bLee et al 2016).…”

“…The baculovirus expression vector system (BEVS) has been proven useful for the production of recombinant proteins in eukaryotic insect cells (Deparis et al 2003;Song et al 2010;Park et al 2011Park et al , 2014Park et al , 2015Lee & Ko 2014;Kim et al 2015;Qiao et al 2015;Lim et al 2015aLim et al , 2015bLee et al 2016). BEVS has many advantages, including scalability using high-density suspension cultures, capacity for expressing multiple genes at high expression levels, and the possibility of post-translational modifications (Deparis et al 2003;Malde & Hunt 2004;Kost et al, 2005).…”

“…Optimization of infection conditions influences the expression of recombinant proteins, such as the monoclonal antibody CO17-1A (Park et al 2011), which recognizes the colorectal antigen GA733-2 (gastrointestinal tumorassociated antigen 2) (Strassburg et al 1992;Zaloudik et al 2002;Lu et al 2012;Kim et al 2015;Park et al 2015;Qiao et al 2015;Lim et al 2015aLim et al , 2015bLee et al 2016). Accurate viral stock titration is crucial for determining the multiplicity of infection (MOI) value, which represents the total number of virions added per cell during infection (Neutra et al 1992;Mulvania et al 2004;Janakiraman et al 2006) and ultimately determines recombinant protein expression (Radford et al, 1997).…”