Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 May:557:15-22.
doi: 10.1016/j.virol.2021.01.004. Epub 2021 Feb 9.

Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2

Teodora Djukic  1 , Maja Mladenovic  1 , Dragana Stanic-Vucinic  1 , Jelena Radosavljevic  1 , Katarina Smiljanic  1 , Ljiljana Sabljic  2 , Marija Devic  2 , Danica Cujic  2 , Tamara Vasovic  1 , Ana Simovic  1 , Mirjana Radomirovic  1 , Tanja Cirkovic Velickovic  3
Affiliations

Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2

Teodora Djukic et al. Virology. 2021 May.

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.

Keywords: COVID-19; Prokaryotic expression; Recombinant nucleocapsid protein; SARS-CoV-2; serological assay.

PubMed Disclaimer

Conflict of interest statement

The authors declare no financial or commercial conflict of interest.

Figures

Fig. 1
Fig. 1
Expression and purification of rfNP. (A) SDS-PAGE of total lysates of transfected E. coli BL21(DE3); lane 1 – MW markers, lane 2 – without induction, lane 3 after induction at 25 °C during 18 h, lane 4 - after induction at 37 °C during 18 h; (B) Western blot of E. coli expressed rfNP probed with anti-His antibodies; (C) frNP purification on Ni Sepharose 6 fast Flow; lane 1 – MW markers, lane 2 - soluble fraction of cell lysates; lane 3- flowthrough, lane 4 – unbound proteins; raction of unbound proteins; lanes 5–10 fractions eluated with imidayole: 50 mM (4),100 mM (5), 200 mM (6)), 250 mM (7), 300 mM (8) and 500 nm (9). (D) rfNP after second purification step by cation exchange chromatography on SP Sepharose; line 1 - MW markers, Line 2 - purified rfNP.
Fig. 2
Fig. 2
Structural characterization of rfNP. Far-UV CD spectra (A) and the secondary structure content (B) of purified rfNP.
Fig. 3
Fig. 3
Sequence coverage of N protein, obtained after mass spectrometry analysis of purified frNP; The inset presents preparative SDS PAGE of purified rfNP with marked 40 kDa band excised for in-gel digestion and MS analysis.
Fig. 4
Fig. 4
Representative immunoblot of rfNP with sera from COVID-19 patients (samples 4,5,8) and healthy individuals (samples 1,2,3,7); N – nonspecific binding of anti-human IgM and IgG antibodies.
Fig. 5
Fig. 5
Distribution of the OD values obtained from 68 SARS-CoV-2 negative and 50 SARS-CoV-2 positive human sera sample using rfNP based SARS-CoV-2 IgG (A) and IgM (B) indirect ELISA. The ROC curve built for 68 SARS-CoV-2 negative and 50 SARS-CoV-2 positive human sera analyzed by nucleocapsid based SARS-CoV-2 IgG (C) and IgM (D)ELISA.
See this image and copyright information in PMC

References

    1. Burbelo P.D., Riedo F.X., Morishima C., Rawlings S., Smith D., Das S., Strich J.R., Chertow D.S., Davey R.T., Cohen J.I. Sensitivity in detection of antibodies to nucleocapsid and spike proteins of severe acute respiratory syndrome coronavirus 2 in patients with coronavirus disease 2019. J. Infect. Dis. 2020;222:206–213. doi: 10.1093/infdis/jiaa273. - DOI - PMC - PubMed
    1. Chu D.K.W., Pan Y., Cheng S.M.S., Hui K.P.Y., Krishnan P., Liu Y., Ng D.Y.M., Wan C.K.C., Yang P., Wang Q., Peiris M., Poon L.L.M. Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia. Clin. Chem. 2020;7:1–7. doi: 10.1093/clinchem/hvaa029. - DOI - PMC - PubMed
    1. Cubuk J., Alston J.J., Incicco J.J., Singh S., Stuchell-Brereton M.D., Ward M.D., Zimmerman M.I., Vithani N., Griffith D., Wagoner J.A., Bowman G.R., Hall K.B., Soranno A., Holehouse A.S. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. bioRxiv. 2020;2:2020. doi: 10.1101/2020.06.17.158121. - DOI - PMC - PubMed
    1. Gorbalenya A.E., Baker S.C., Baric R.S., de Groot R.J., Drosten C., Gulyaeva A.A., Haagmans B.L., Lauber C., Leontovich A.M., Neuman B.W., Penzar D., Perlman S., Poon L.L.M., Samborskiy D.V., Sidorov I.A., Sola I., Ziebuhr J. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat. Microbiol. 2020;5:536–544. doi: 10.1038/s41564-020-0695-z. - DOI - PMC - PubMed
    1. Jiang H. wei, Li Y., Zhang H. nan, Wang W., Yang X., Qi H., Li H., Men D., Zhou J., Tao S. ce. SARS-CoV-2 proteome microarray for global profiling of COVID-19 specific IgG and IgM responses. Nat. Commun. 2020;11:1–11. doi: 10.1038/s41467-020-17488-8. - DOI - PMC - PubMed

Publication types

MeSH terms