Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells
2023
Authors:
Sibinčić, NikolinaKrstić Ristivojević, Maja
Stojanović, Marijana
Mladenović Stokanić, Maja
Vasović, Tamara
Ćirković Veličković, Tanja
Stojadinović, Marija
Contributors
Spasojević, IvanDocument Type:
Conference object (Published version)
,
© 2023 by the Faculty of Chemistry, University of Belgrade
Metadata
Show full item recordAbstract:
The SARS-CoV-2 nucleocapsid (N) protein plays a significant role in the coronavirus life cycle and participates in a variety of critical events following viral invasion1. In infected patients, high titers of immunoglobulin G (IgG) targeting N protein were detected and correlated with the clinical course of the disease2. Therefore, N protein and anti-N protein IgGs were recognized as important diagnostic indicators of COVID-19 infection in serological and quick antigen tests3. In this study, we optimized the expression of the recombinant form of SARS-CoV-2 N protein in a mammalian cell line HEK293T by comparing the transfection efficiency between Polyethylenimine (PEI) and Calcium Phosphate (CaP) DNA-complexing agents. Transfection potency was tested at different cell confluence and passage number, in several cell culture media, pre-transfection and post-transfection media change and in conditions of reduced serum. Chloroquine and glycerol treatments were included to enhance transfection efficiency as they might inhibit DNA degradation in lysosomes or increase membrane permeability. Protein expression was monitored in cell supernatants up to 7 days post-transfection in dot-bot and Western blot using anti-N protein antibodies. Both transfection methods have shown moderate to relatively high transfection efficiency dependent on the applied conditions, making them affordable and easy to use techniques for recombinant N protein production on a small-scale in adherent mammalian systems. PEI acts as a good delivery system regardless of the presence of the fetal bovine serum (FBS), while CaP transfection is more dependent on the presence of FBS which in turn favors N protein degradation. However, we have optimized both methods to achieve optimal expression of unfragmented N-protein in serum-free conditions. Apart from setting up a cost-effective platform for expression of N protein in mammalian cells, we plan on investigating the mechanisms behind the PEI and CaP non-viral gene delivery systems as there are still some uncertainties in the scientific community.
Funding / projects:
- Ministry of Science, Technological Development and Innovation of the Republic of Serbia, institutional funding - 200168 (University of Belgrade, Faculty of Chemistry) (RS-MESTD-inst-2020-200168)
- Ministry of Science, Technological Development and Innovation of the Republic of Serbia, institutional funding - 200288 (Innovation Center of the Faculty of Chemistry) (RS-MESTD-inst-2020-200288)
- CAPSIDO – Developement of the assays for detection of SARS Cov-2 virus capsid proteins in biological fluids of COVID19 patients (RS-ScienceFundRS-Fond_2020_COVID19-7542203)
In:
- Spasojević I, editor. Biochemistry in Biotechnology: Serbian Biochemical Society, Twelfth Conference, International scientific meeting; 2023 Sep 21-23; Belgrade, Serbia. Belgrade: Faculty of Chemistry; 2023. p. 91-2.