High level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilization

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dc.contributor.authorCHURA-CHAMBI, ROSA M.pt_BR
dc.contributor.authorPRIETO-DA-SILVA, ALVARO R. de B.pt_BR
dc.contributor.authorDI LELA, MATHEUS M.pt_BR
dc.contributor.authorOLIVEIRA, JOAO E.pt_BR
dc.contributor.authorABREU, PATRICIA E.A.pt_BR
dc.contributor.authorMEIRELES, LUCIANA R.pt_BR
dc.contributor.authorANDRADE JUNIOR, HEITOR F. dept_BR
dc.contributor.authorMORGANTI, LIGIApt_BR
dc.coverageInternacionalpt_BR
dc.date.accessioned2022-05-04T11:49:55Z
dc.date.available2022-05-04T11:49:55Z
dc.date.issued2022pt_BR
dc.description.abstractSARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.pt_BR
dc.format.extent1-14pt_BR
dc.identifier.citationCHURA-CHAMBI, ROSA M.; PRIETO-DA-SILVA, ALVARO R. de B.; DI LELA, MATHEUS M.; OLIVEIRA, JOAO E.; ABREU, PATRICIA E.A.; MEIRELES, LUCIANA R.; ANDRADE JUNIOR, HEITOR F. de; MORGANTI, LIGIA. High level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilization: application of high pressure at pH 9.0. <b>PLoS One</b>, v. 17, n. 2, p. 1-14, 2022. DOI: <a href="https://dx.doi.org/10.1371/journal.pone.0262591">10.1371/journal.pone.0262591</a>. Disponível em: http://repositorio.ipen.br/handle/123456789/33024.
dc.identifier.doi10.1371/journal.pone.0262591pt_BR
dc.identifier.fasciculo2pt_BR
dc.identifier.issn1932-6203pt_BR
dc.identifier.orcid0000-0002-6937-1120pt_BR
dc.identifier.orcidhttps://orcid.org/0000-0002-7870-1793
dc.identifier.percentilfi65.1pt_BR
dc.identifier.percentilfiCiteScore87pt_BR
dc.identifier.urihttp://repositorio.ipen.br/handle/123456789/33024
dc.identifier.vol17pt_BR
dc.relation.ispartofPLoS Onept_BR
dc.rightsopenAccesspt_BR
dc.subjectcoronaviruses
dc.subjectproteins
dc.subjectantibodies
dc.subjectenzyme immunoassay
dc.subjectpressure range mega pa 10-100
dc.titleHigh level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilizationpt_BR
dc.typeArtigo de periódicopt_BR
dspace.entity.typePublication
ipen.autorLIGIA ELY MORGANTI FERREIRA DIAS
ipen.autorJOAO EZEQUIEL DE OLIVEIRA
ipen.autorMATHEUS MARTINS DI LELA
ipen.autorROSA MARIA CHURA CHAMBI
ipen.codigoautor296
ipen.codigoautor425
ipen.codigoautor15210
ipen.codigoautor3338
ipen.contributor.ipenauthorLIGIA ELY MORGANTI FERREIRA DIAS
ipen.contributor.ipenauthorJOAO EZEQUIEL DE OLIVEIRA
ipen.contributor.ipenauthorMATHEUS MARTINS DI LELA
ipen.contributor.ipenauthorROSA MARIA CHURA CHAMBI
ipen.date.recebimento22-05
ipen.identifier.fi3.7pt_BR
ipen.identifier.fiCiteScore6pt_BR
ipen.identifier.ipendoc28686pt_BR
ipen.identifier.iwosWoSpt_BR
ipen.identifier.ods3
ipen.range.fi3.000 - 4.499
ipen.range.percentilfi50.00 - 74.99
ipen.subtituloapplication of high pressure at pH 9.0pt_BR
ipen.type.genreArtigo
relation.isAuthorOfPublicationc26a78ae-be8e-4a8d-b22c-0a62eb422f8b
relation.isAuthorOfPublicatione9e6346c-9b8b-4d39-8bdd-505ea6997646
relation.isAuthorOfPublication4e6bbeb0-e69c-437f-86ff-ba0c68310564
relation.isAuthorOfPublicationd2b97487-e004-4056-8df7-0ab5c8d5cacc
relation.isAuthorOfPublication.latestForDiscoveryd2b97487-e004-4056-8df7-0ab5c8d5cacc
sigepi.autor.atividadeMORGANTI, LIGIA:296:810:Npt_BR
sigepi.autor.atividadeOLIVEIRA, JOAO E.:425:810:Npt_BR
sigepi.autor.atividadeDI LELA, MATHEUS M.:15210:810:Npt_BR
sigepi.autor.atividadeCHURA-CHAMBI, ROSA M.:3338:810:Spt_BR
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