Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production.
SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in
E. coli
in soluble form, purified and characterized biochemically and immunologically.
Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of β-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively).
Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.
Oxidative modifications of proteins are key to many applications in biotechnology. Metal-catalyzed oxidation reactions efficiently oxidize proteins but with low selectivity, and are highly dependent on the protein surface residues to direct the reaction. Herein, we demonstrate that discrete inorganic ligands such as polyoxometalates enable an efficient and selective protein oxidative cleavage. In the presence of ascorbate (1 mM), the Cu-substituted polyoxometalate K8[Cu2+(H2O)(α2-P2W17O61)], (CuIIWD, 0.05 mM) selectively cleave hen egg white lysozyme under physiological conditions (pH =7.5, 37 °C) producing only four bands in the gel electropherogram (12.7, 11, 10, and 5 kDa). Liquid chromatography/mass spectrometry analysis reveals a regioselective cleavage in the vicinity of crystallographic CuIIWD/lysozyme interaction sites. Mechanistically, polyoxometalate is critical to position the Cu at the protein surface and limit the generation of oxidative species to the proximity of binding sites. Ultimately, this study outlines the potential of discrete, designable metal oxo clusters as catalysts for the selective modification of proteins through radical mechanisms under non-denaturing conditions.
In this study, we investigated structural aspects of covalent binding of food derived blue pigment phycocyanobilin (PCB) to bovine β-lactoglobulin (BLG), major whey protein, by spectroscopic, electrophoretic, mass spectrometry and computational methods. At physiological pH (7.2), we found that covalent pigment binding via free cysteine residue is slow (k = 0.065 min), of moderate affinity (K = 4 × 10 M), and stereo-selective. Binding also occurs at a broad pH range and under simulated gastrointestinal conditions. Adduct formation rises with pH, and in concentrated urea (k = 0.101 min). The BLG-PCB adduct has slightly altered secondary and tertiary protein structure, and bound PCB has higher fluorescence and more stretched conformation than free chromophore. Combination of steered molecular dynamic for disulfide exchange, non-covalent and covalent docking, favours Cys119 residue in protein calyx as target for covalent BLG-PCB adduct formation. Our results suggest that this adduct can serve as delivery system of bioactive PCB.
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Figure S1. Optimized structures of two atropisomers of the clozapine molecule. Left: P (plus) atropisomer. Right: M (minus) atropisomer. Two atropisomers are also enantiomers.
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