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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.
De Vos, Jelle; Pereira Aguilar, Patricia; Köppl, Christoph; Fischer, Andreas; Grünwald-Gruber, Clemens; Dürkop, Mark; Klausberger, Miriam; Mairhofer, Juergen; Striedner, Gerald; Cserjan-Puschmann, Monika; Jungbauer, Alois; Lingg, Nico.
  • De Vos J; Vrije Universiteit Brussel, Department of Chemical Engineering, 1050, Brussels, Belgium; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), 1190 Vienna, Austria.
  • Pereira Aguilar P; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), 1190 Vienna, Austria; acib - Austrian Centre of Industrial Biotechnology, 1190, Vienna, Austria. Electronic address: patricia.aguilar@boku.ac.at.
  • Köppl C; acib - Austrian Centre of Industrial Biotechnology, 1190, Vienna, Austria.
  • Fischer A; acib - Austrian Centre of Industrial Biotechnology, 1190, Vienna, Austria.
  • Grünwald-Gruber C; BOKU Core Facility Mass Spectrometry, University of Natural Resources and Life Sciences, Vienna (BOKU), 1190, Vienna, Austria.
  • Dürkop M; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), 1190 Vienna, Austria; Novasign GmbH, 1190, Vienna, Austria.
  • Klausberger M; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), 1190 Vienna, Austria.
  • Mairhofer J; enGenes Biotech GmbH, 1190, Vienna, Austria.
  • Striedner G; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), 1190 Vienna, Austria; acib - Austrian Centre of Industrial Biotechnology, 1190, Vienna, Austria; enGenes Biotech GmbH, 1190, Vienna, Austria.
  • Cserjan-Puschmann M; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), 1190 Vienna, Austria; acib - Austrian Centre of Industrial Biotechnology, 1190, Vienna, Austria.
  • Jungbauer A; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), 1190 Vienna, Austria; acib - Austrian Centre of Industrial Biotechnology, 1190, Vienna, Austria.
  • Lingg N; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), 1190 Vienna, Austria; acib - Austrian Centre of Industrial Biotechnology, 1190, Vienna, Austria.
Talanta ; 235: 122691, 2021 Dec 01.
Article in English | MEDLINE | ID: covidwho-1313446
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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Nucleocapsid Proteins / COVID-19 Limits: Humans Language: English Journal: Talanta Year: 2021 Document Type: Article Affiliation country: J.talanta.2021.122691

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Nucleocapsid Proteins / COVID-19 Limits: Humans Language: English Journal: Talanta Year: 2021 Document Type: Article Affiliation country: J.talanta.2021.122691