Transient Expression in HEK-293 Cells in Suspension Culture as a Rapid and Powerful Tool: SARS-CoV-2 N and Chimeric SARS-CoV-2N-CD154 Proteins as a Case Study.

Publication date: Nov 14, 2023

In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein.

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Concepts Keywords
Bioreactors HEK-293
Cd40 N protein
Diminish SARS-CoV-2
Ligand serum-free
Vaccines suspension culture
transient expression


Type Source Name
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disease VO vaccine antigen
disease VO immunization
disease VO time
disease VO Canada
disease IDO process
disease MESH covid19
disease VO Betacoronavirus
drug DRUGBANK Coenzyme M
pathway REACTOME Translation
drug DRUGBANK Spinosad
disease IDO production
drug DRUGBANK Sodium lauryl sulfate
disease VO USA
drug DRUGBANK Glutamic Acid
drug DRUGBANK Streptomycin
drug DRUGBANK Histidine
disease VO viability
disease IDO cell
disease VO viable
disease VO volume
drug DRUGBANK Oxygen
drug DRUGBANK Medical air
disease VO injection
drug DRUGBANK Sodium bicarbonate
drug DRUGBANK Sulodexide
drug DRUGBANK Imidazole
drug DRUGBANK Methylergometrine
disease VO storage
drug DRUGBANK Sucrose
drug DRUGBANK L-Leucine
drug DRUGBANK Albendazole
drug DRUGBANK Aspartame
disease MESH death
disease IDO quality
disease VO efficiency
disease VO effective
disease VO Glycoprotein
disease VO vaccine candidate
drug DRUGBANK Diethylstilbestrol
drug DRUGBANK Erythropoietin
drug DRUGBANK (S)-Des-Me-Ampa
disease MESH convalescence
drug DRUGBANK Guanosine
disease VO efficient
disease IDO assay
disease MESH infections
drug DRUGBANK Carboxyamidotriazole
disease VO vaccination
disease VO titer

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