Native Hydrophobic Interaction Chromatography Hyphenated to Multi-Angle Light Scattering Detection for In-Process Control of SARS-CoV-2 Nucleocapsid Protein Produced in Escherichia Coli

11 March 2021, Version 1


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 to dimerization and interaction with nucleic acids. A native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS) method was established for in-process control, in particular, to monitor product fragmentation and multimerization throughout the purification process. High titers of the nucleocapsid protein 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 chromatographic steps. 730 mg purified NP per liter of fermentation could be produced by the optimized process, corresponding to a yield of 77%. 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 HIC-MALS method enables to monitor the purity of the product and simultaneously access its molecular mass.


2019 novel coronavirus
Hydrophobic interaction chromatography
Downstream processing
CASPON technology

Supplementary materials

NP SI 2021


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