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- W3185956999 abstract "The nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for several steps of the viral life cycle, and is abundantly expressed during infection, making it an ideal diagnostic target protein. This protein has a strong tendency for dimerization and interaction with nucleic acids. For the first time, high titers of NP were expressed in E. coli with a CASPON tag, using a growth-decoupled protein expression system. Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of downstream processing (DSP) steps. An analytical method consisting of native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS) was established for in-process control, in particular, to monitor product fragmentation and multimerization throughout the purification process. 730 mg purified NP per liter of fermentation could be produced by the optimized process, corresponding to a yield of 77% after cell lysis. The HIC-MALS method was used to demonstrate that the NP product can be produced with a purity of 95%. The molecular mass of the main NP fraction is consistent with dimerized protein as was verified by a complementary native size-exclusion separation (SEC)-MALS analysis. Peptide mapping mass spectrometry and host cell specific enzyme-linked immunosorbent assay confirmed the high product purity, and the presence of a minor endogenous chaperone explained the residual impurities. The optimized HIC-MALS method enables monitoring of the product purity, and simultaneously access its molecular mass, providing orthogonal information complementary to established SEC-MALS methods. Enhanced resolving power can be achieved over SEC, attributed to the extended variables to tune selectivity in HIC mode." @default.
- W3185956999 created "2021-08-02" @default.
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- W3185956999 date "2021-12-01" @default.
- W3185956999 modified "2023-10-18" @default.
- W3185956999 title "Production of full-length SARS-CoV-2 nucleocapsid protein from Escherichia coli optimized by native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection" @default.
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- W3185956999 doi "https://doi.org/10.1016/j.talanta.2021.122691" @default.
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