Citation
- Authors: Lavado-García J. et al.
- Year: 2022
- Journal: Biotechnol Bioeng
- Applications: in vitro / DNA / PEIpro
- Cell type: HEK-293
Description: Human embryonic kidney Fibroblast
Known as: HEK293, 293
Method
Transfections were carried out at a cell density of 2x106 cells/mL using a final DNA concentration of 1 µg/mL. PEI/DNA complexes were formed by adding PEI to plasmid DNA diluted in fresh culture medium (10% of the total culture volume to be transfected). Transfection reagent PEIpro® (Polyplus transfection) was used.
Briefly, the corresponding plasmid was diluted with supplemented HyCell™ culture media and vortexed for 10 s. Then PEI was added in 1:2 (w/w) DNA:PEI ratio and vortexed three times, incubated for 15 min at room temperature (RT) and then added to the cell culture. The plasmid used encoded a HIV-Gag protein fused in frame to eGFP (Gag::eGFP). As a transfection control, a plasmid sharing the same backbone but lacking the Gag::eGFP gene was used and noted as mock.
Abstract
HIV-1 virus-like particles (VLPs) are nanostructures derived from the self-assembly and cell budding of Gag polyprotein. Mimicking the native structure of the virus and being non-infectious, they represent promising candidates for the development of new vaccines as they elicit a strong immune response. In addition to this, the bounding membrane can be functionalized with exogenous antigens to target different diseases. Protein glycosylation depends strictly on the production platform and expression system used and the displayed glycosylation patterns may influence down-stream processing as well as the immune response. One of the main challenges for the development of Gag VLP production bioprocess is the separation of VLPs and coproduced extracellular vesicles (EVs). In this work, porous graphitized carbon separation method coupled with mass spectrometry was used to characterize the N- and O- glycosylation profiles of Gag VLPs produced in HEK293 cells. We identified differential glycan signatures between VLPs and EVs that could pave the way for further separation and purification strategies in order to optimize downstream processing and move forward in VLP-based vaccine production technology. This article is protected by copyright. All rights reserved.