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

Abstract

<p>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. <a>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 <i>E. coli</i> with a CASPON tag, using a growth-decoupled protein expression system<i>. </i>Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of chromatographic steps</a>. 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.</p>

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests

“…The remaining impurities consisted of NP-related fragments and RNA. Residual host cell protein concentration was 0.9 ng/mg NP and dsDNA concentration was 1 mg/ mg NP, as determined by De Vos and colleagues [10]. NP has an intrinsic propensity to oligomerize and displays very slow dissociation from the NP-specific antibody (Abcam, ab272852).…”

“…A pET30acer E. coli expression vector [7] encoding the full-length SARS-CoV-2 Wuhan-1 NP sequence (aa Met1ÀAla419, GenBank: NC_045512.2) [5] fused to a completely removable N-terminal CAS-PON tag [8,9], yielding pET30acer-CASPONÀ ÀNP, was generated as described elsewhere [10]. Briefly, SARS-CoV-2 NP sequence was amplified via PCR using the qPCR positive control plasmid 2019-nCoV_N obtained from Integrated DNA Technologies (Coralville, Iowa, USA) and was fused to the CASPON tag consisting of the negative charged T7AC solubility tag [8], a hexa-histidine tag, a short linker (GSG) and the caspase-2 cleavage site (VDVAD) resulting in the sequence MLEDPERNKERKEAELQAQTAEQHHHHHHGSGVDVAD.…”

“…Purification of SARS-CoV-2 NP from E. coli cellular lysates: The purification of NP was optimised and performed as described by De Vos and colleagues [10]. In brief, NP was produced by using the CASPON platform process [9] with modifications.…”