Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

Characterization of the complex formation of 1,6-anhydro-ß-maltose and potassium ions using NMR spectroscopy and single-crystal X-ray crystallography.

The formation of a complex between 1,6-anhydro-ß-maltose and potassium ions was characterized using (1)H, (13)C and (39)K NMR spectroscopy and single-crystal X-ray crystallography. In the NMR study, the spin-lattice relaxation times (T(1)) of C1, C3, C5, C6, and C5' significantly decreased in the presence of potassium ions, and (39)K-T(1) also decreased in the presence of 1,6-anhydro-ß-maltose, indicating complex formation. In a crystal, both 8- and 9-coordination structures, corresponding to the distorted capped pentagonal bipyramidal structure and the capped hexagonal bipyramidal structure, respectively, were identified. A potassium ion was positioned in the center of each bipyramidal structure.

Characterization of the complex formation of 1,6-anhydro-beta-maltotriose with potassium using 1H and 39K NMR spectroscopy.

The (1)H and (39)K longitudinal relaxation times (T(1)) and (1)H diffusion coefficients were measured to investigate the complex formation of 1,6-anhydro-beta-maltotriose and potassium ions. Although the (1)H-T(1) values of H3', H5', H1'' and H4'' decreased in the presence of potassium ions, (1)H chemical shifts and (1)H diffusion coefficients did not show significant changes. The long-range coupling constants of (3)J(C-H) around the glycosyl bonds did not show significant changes either. In the measurements of (39)K spectra, the (39)K signal obviously broadened and the (39)K-T(1) values decreased in the presence of 1,6-anhydro-beta-maltotriose, indicating the complex formation of 1,6-anhydro-beta-maltotriose and potassium ions. These results indicate that the conformation and molecular volume were unaffected in the complex formation.