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  - JOUR
AU  - Platanić-Arizanović, Lena
AU  - Gligorijević, Nikola
AU  - Cvijetić, Ilija
AU  - Mijatović, Aleksandar
AU  - Krstić-Ristivojević, Maja
AU  - Minić, Simeon
AU  - Nikolić-Kokić, Aleksandra
AU  - Miljević, Čedo
AU  - Nikolić, Milan
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6028
AB  - : Packed with hemoglobin, an essential protein for oxygen transport, human erythrocytes
are a suitable model system for testing the pleiotropic effects of lipophilic drugs. Our study investigated the interaction between antipsychotic drugs clozapine, ziprasidone, sertindole, and human
hemoglobin under simulated physiological conditions. Analysis of protein fluorescence quenching
at different temperatures and data obtained from the van’t Hoff diagram and molecular docking
indicate that the interactions are static and that the tetrameric human hemoglobin has one binding site
for all drugs in the central cavity near αβ interfaces and is dominantly mediated through hydrophobic
forces. The association constants were lower-moderate strength (~104 M−1
), the highest observed
for clozapine (2.2 × 104 M−1 at 25 ◦C). The clozapine binding showed “friendly” effects: increased
α-helical content, a higher melting point, and protein protection from free radical-mediated oxidation.
On the other hand, bound ziprasidone and sertindole had a slightly pro-oxidative effect, increasing
ferrihemoglobin content, a possible “foe”. Since the interaction of proteins with drugs plays a vital
role in their pharmacokinetic and pharmacodynamic properties, the physiological significance of the
obtained findings is briefly discussed.
PB  - Basel: MDPI
T2  - International Journal of Molecular Sciences
T1  - Human Hemoglobin and Antipsychotics Clozapine, Ziprasidone and Sertindole: Friends or Foes?
IS  - 10
VL  - 24
DO  - 10.3390/ijms24108921
SP  - 8921
ER  - 

@article{
author = "Platanić-Arizanović, Lena and Gligorijević, Nikola and Cvijetić, Ilija and Mijatović, Aleksandar and Krstić-Ristivojević, Maja and Minić, Simeon and Nikolić-Kokić, Aleksandra and Miljević, Čedo and Nikolić, Milan",
year = "2023",
abstract = ": Packed with hemoglobin, an essential protein for oxygen transport, human erythrocytes
are a suitable model system for testing the pleiotropic effects of lipophilic drugs. Our study investigated the interaction between antipsychotic drugs clozapine, ziprasidone, sertindole, and human
hemoglobin under simulated physiological conditions. Analysis of protein fluorescence quenching
at different temperatures and data obtained from the van’t Hoff diagram and molecular docking
indicate that the interactions are static and that the tetrameric human hemoglobin has one binding site
for all drugs in the central cavity near αβ interfaces and is dominantly mediated through hydrophobic
forces. The association constants were lower-moderate strength (~104 M−1
), the highest observed
for clozapine (2.2 × 104 M−1 at 25 ◦C). The clozapine binding showed “friendly” effects: increased
α-helical content, a higher melting point, and protein protection from free radical-mediated oxidation.
On the other hand, bound ziprasidone and sertindole had a slightly pro-oxidative effect, increasing
ferrihemoglobin content, a possible “foe”. Since the interaction of proteins with drugs plays a vital
role in their pharmacokinetic and pharmacodynamic properties, the physiological significance of the
obtained findings is briefly discussed.",
publisher = "Basel: MDPI",
journal = "International Journal of Molecular Sciences",
title = "Human Hemoglobin and Antipsychotics Clozapine, Ziprasidone and Sertindole: Friends or Foes?",
number = "10",
volume = "24",
doi = "10.3390/ijms24108921",
pages = "8921"
}

Platanić-Arizanović, L., Gligorijević, N., Cvijetić, I., Mijatović, A., Krstić-Ristivojević, M., Minić, S., Nikolić-Kokić, A., Miljević, Č.,& Nikolić, M.. (2023). Human Hemoglobin and Antipsychotics Clozapine, Ziprasidone and Sertindole: Friends or Foes?. in International Journal of Molecular Sciences
Basel: MDPI., 24(10), 8921.
https://doi.org/10.3390/ijms24108921

Platanić-Arizanović L, Gligorijević N, Cvijetić I, Mijatović A, Krstić-Ristivojević M, Minić S, Nikolić-Kokić A, Miljević Č, Nikolić M. Human Hemoglobin and Antipsychotics Clozapine, Ziprasidone and Sertindole: Friends or Foes?. in International Journal of Molecular Sciences. 2023;24(10):8921.
doi:10.3390/ijms24108921 .

Platanić-Arizanović, Lena, Gligorijević, Nikola, Cvijetić, Ilija, Mijatović, Aleksandar, Krstić-Ristivojević, Maja, Minić, Simeon, Nikolić-Kokić, Aleksandra, Miljević, Čedo, Nikolić, Milan, "Human Hemoglobin and Antipsychotics Clozapine, Ziprasidone and Sertindole: Friends or Foes?" in International Journal of Molecular Sciences, 24, no. 10 (2023):8921,
https://doi.org/10.3390/ijms24108921 . .