Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2
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2021
Authors
Đukić, TeodoraMladenović, Maja
Stanić-Vučinić, Dragana

Radosavljević, Jelena

Smiljanić, Katarina

Sabljić, Ljiljana
Dević, Marija
Ćujić, Danica
Vasović, Tamara
Simović, Ana

Radomirović, Mirjana Ž.

Ćirković-Veličković, Tanja

Article (Submitted Version)
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Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production.
SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically.
Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of β-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively).
Cost-effective approach for soluble recombinant N protein fragment... production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.
Keywords:
Recombinant nucleocapsid protein / COVID-19 / SARS-CoV-2 / Prokaryotic expression / serological assaySource:
Virology journal, 2021, 557, 15-22Publisher:
- Elsevier
Projects:
- CAPSIDO – Developement of the assays for detection of SARS Cov-2 virus capsid proteins in biological fluids of COVID19 patients (RS-7542203)
- Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 451-03-68/2020-14/200168 (University of Belgrade, Faculty of Chemistry) (RS-200168)
- Serbian Academy of Sciences and Arts GA No. F-26.
- UNDP project "Preventing and Responding to COVID-19 in At-risk areas - Sustainable production of the serological IgG test for SARS CoV-2 in Serbia".
Note:
- This is the preprint of the article: Đukić, T.; Mladenović, M.; Stanić-Vučinić, D.; Radosavljević, J.; Smiljanić, K.; Sabljić, L.; Dević, M.; Ćujić, D.; Vasović, T.; Simović, A.; Radomirović, M. Ž.; Ćirković-Veličković, T. Expression, Purification and Immunological Characterization of Recombinant Nucleocapsid Protein Fragment from SARS-CoV-2. Virology journal 2021, 557, 15–22. https://doi.org/10.1016/j.virol.2021.01.004.
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Hemijski fakultetTY - JOUR AU - Đukić, Teodora AU - Mladenović, Maja AU - Stanić-Vučinić, Dragana AU - Radosavljević, Jelena AU - Smiljanić, Katarina AU - Sabljić, Ljiljana AU - Dević, Marija AU - Ćujić, Danica AU - Vasović, Tamara AU - Simović, Ana AU - Radomirović, Mirjana Ž. AU - Ćirković-Veličković, Tanja PY - 2021 UR - http://cherry.chem.bg.ac.rs/handle/123456789/4330 AB - Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of β-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection. PB - Elsevier T2 - Virology journal T1 - Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2 VL - 557 SP - 15 EP - 22 DO - 10.1016/j.virol.2021.01.004 ER -
@article{ author = "Đukić, Teodora and Mladenović, Maja and Stanić-Vučinić, Dragana and Radosavljević, Jelena and Smiljanić, Katarina and Sabljić, Ljiljana and Dević, Marija and Ćujić, Danica and Vasović, Tamara and Simović, Ana and Radomirović, Mirjana Ž. and Ćirković-Veličković, Tanja", year = "2021", url = "http://cherry.chem.bg.ac.rs/handle/123456789/4330", abstract = "Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of β-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.", publisher = "Elsevier", journal = "Virology journal", title = "Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2", volume = "557", pages = "15-22", doi = "10.1016/j.virol.2021.01.004" }
Đukić T, Mladenović M, Stanić-Vučinić D, Radosavljević J, Smiljanić K, Sabljić L, Dević M, Ćujić D, Vasović T, Simović A, Radomirović MŽ, Ćirković-Veličković T. Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2. Virology journal. 2021;557:15-22
Đukić, T., Mladenović, M., Stanić-Vučinić, D., Radosavljević, J., Smiljanić, K., Sabljić, L., Dević, M., Ćujić, D., Vasović, T., Simović, A., Radomirović, M. Ž.,& Ćirković-Veličković, T. (2021). Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2. Virology journalElsevier., 557, 15-22. https://doi.org/10.1016/j.virol.2021.01.004
Đukić Teodora, Mladenović Maja, Stanić-Vučinić Dragana, Radosavljević Jelena, Smiljanić Katarina, Sabljić Ljiljana, Dević Marija, Ćujić Danica, Vasović Tamara, Simović Ana, Radomirović Mirjana Ž., Ćirković-Veličković Tanja, "Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2" 557 (2021):15-22, https://doi.org/10.1016/j.virol.2021.01.004 .