• Authors: Samuelsson E. et al.
  • Year: 2022
  • Journal: ACS Infect Dis 8 1883-1893
  • Applications: in vitro / DNA / FectoCHO Expression System, FectoPRO
  • Cell types:
    1. Name: CHO-S
      Description: Chinese hamster ovary cells
    2. Name: FreeStyle 293-F
    3. Name: Lec3.2.8.1


Expression of recombinant S protein constructs The receptor-binding domain of the SARS-CoV-2 spike protein (amino acids 319-541) was produced in three cell lines using an expression vector obtained through BEI Resources, NIAID, NIH, which is vector pCAGGS containing the SARS-CoV-2, Wuhan-Hu-1 spike glycoprotein gene RBD with C-terminal Hexa-Histidine tag (NR-52309). The HEK293 derivate HEK293F cell line (Cat nr R79007, Thermo Fisher Scientific) were cultured in Freestyle 293 medium. Cells were transfected at 2 × 106 cells/mL using FectoPro transfection reagent (Polyplus transfection). Transfected HEK293F cells were kept at 37 °C. Protein-containing culture supernatants (800 mL – 1 L) were harvested when cell viability was below 80 %, which was after 90 hours (HEK293F), filtered using Polydisc AS 0.45 μm (Whatman, Maidstone, UK) and loaded onto a 5 mL HisExcel column (Cytiva, Marlborough, MA). After sample loading, the column was washed with 20 mM sodium phosphate, 0.5 M NaCl and 30 mM imidazole before elution of the protein using the same buffer with 300 mM imidazole (HEK293F-produced RBD). Pooled fractions were concentrated using 10 kDa Vivaspin concentrators (MWCO 10 kDa, Sartorius, Göttingen, Germany), passed over a HiPrep 26/10 desalting column (Cytiva) in phosphate-buffered saline and finally concentrated again.


The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of the recombinant RBD produced in different mammalian cells. We found a higher degree of complex-type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated, and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity, while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity, verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it is possible to enhance the immunogenicity of recombinant viral proteins.