TY  - CONF
AU  - Sibinčić, Nikolina
AU  - Krstić Ristivojević, Maja
AU  - Stojanović, Marijana
AU  - Mladenović Stokanić, Maja
AU  - Vasović, Tamara
AU  - Ćirković Veličković, Tanja
AU  - Stojadinović, Marija
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6318
AB  - 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.
PB  - Belgrade: Faculty of Chemistry
C3  - Biochemistry in Biotechnology: Serbian Biochemical Society, Twelfth Conference, International scientific meeting; 2023 Sep 21-23; Belgrade, Serbia
T1  - Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells
SP  - 91
EP  - 92
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6318
ER  - 

@conference{
author = "Sibinčić, Nikolina and Krstić Ristivojević, Maja and Stojanović, Marijana and Mladenović Stokanić, Maja and Vasović, Tamara and Ćirković Veličković, Tanja and Stojadinović, Marija",
year = "2023",
abstract = "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.",
publisher = "Belgrade: Faculty of Chemistry",
journal = "Biochemistry in Biotechnology: Serbian Biochemical Society, Twelfth Conference, International scientific meeting; 2023 Sep 21-23; Belgrade, Serbia",
title = "Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells",
pages = "91-92",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6318"
}

Sibinčić, N., Krstić Ristivojević, M., Stojanović, M., Mladenović Stokanić, M., Vasović, T., Ćirković Veličković, T.,& Stojadinović, M.. (2023). Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells. in Biochemistry in Biotechnology: Serbian Biochemical Society, Twelfth Conference, International scientific meeting; 2023 Sep 21-23; Belgrade, Serbia
Belgrade: Faculty of Chemistry., 91-92.
https://hdl.handle.net/21.15107/rcub_ibiss_6318

Sibinčić N, Krstić Ristivojević M, Stojanović M, Mladenović Stokanić M, Vasović T, Ćirković Veličković T, Stojadinović M. Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells. in Biochemistry in Biotechnology: Serbian Biochemical Society, Twelfth Conference, International scientific meeting; 2023 Sep 21-23; Belgrade, Serbia. 2023;:91-92.
https://hdl.handle.net/21.15107/rcub_ibiss_6318 .

Sibinčić, Nikolina, Krstić Ristivojević, Maja, Stojanović, Marijana, Mladenović Stokanić, Maja, Vasović, Tamara, Ćirković Veličković, Tanja, Stojadinović, Marija, "Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells" in Biochemistry in Biotechnology: Serbian Biochemical Society, Twelfth Conference, International scientific meeting; 2023 Sep 21-23; Belgrade, Serbia (2023):91-92,
https://hdl.handle.net/21.15107/rcub_ibiss_6318 .

TY  - GEN
AU  - Ćirković Veličković, Tanja
AU  - Simić, Tatjana
AU  - Dinić, Svetlana
PY  - 2016
UR  - http://www.scopus.com/inward/record.url?eid=2-s2.0-84966388970&partnerID=tZOtx3y1
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/2530
T2  - EuPA Open Proteomics
T1  - The Serbian Proteomics Association (SePA)
VL  - 11
DO  - 10.1016/j.euprot.2016.03.013
SP  - 39
EP  - 40
ER  - 

@misc{
author = "Ćirković Veličković, Tanja and Simić, Tatjana and Dinić, Svetlana",
year = "2016",
journal = "EuPA Open Proteomics",
title = "The Serbian Proteomics Association (SePA)",
volume = "11",
doi = "10.1016/j.euprot.2016.03.013",
pages = "39-40"
}

Ćirković Veličković, T., Simić, T.,& Dinić, S.. (2016). The Serbian Proteomics Association (SePA). in EuPA Open Proteomics, 11, 39-40.
https://doi.org/10.1016/j.euprot.2016.03.013

Ćirković Veličković T, Simić T, Dinić S. The Serbian Proteomics Association (SePA). in EuPA Open Proteomics. 2016;11:39-40.
doi:10.1016/j.euprot.2016.03.013 .

Ćirković Veličković, Tanja, Simić, Tatjana, Dinić, Svetlana, "The Serbian Proteomics Association (SePA)" in EuPA Open Proteomics, 11 (2016):39-40,
https://doi.org/10.1016/j.euprot.2016.03.013 . .