High level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilization: Application of high pressure at pH 9.0


Publication type
Article
Language
English
Access rights
Open access
Terms of use
CC BY
Appears in Collections:
Metrics
Abstract
SARS-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.
Reference
Chura-Chambi RM, Prieto da Silva ARB, Lela MMD, Oliveira JE, Abreu PAE, Meireles LR, et al. High level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilization: Application of high pressure at pH 9.0. PloS One. 2022 Feb;17(2):e0262591. doi:10.1371/journal.pone.0262591.
Link to cite this reference
https://repositorio.butantan.gov.br/handle/butantan/4140
Journal title
Issue Date
2022


Files in This Item:

journal.pone.0262591.pdf
Description:
Size: 2.59 MB
Format: Adobe PDF
View/Open
Show full item record

This item is licensed under a Creative Commons License Creative Commons