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- Title
Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).
- Authors
Tu, Thao; Rathnayaka, Tharangani; Kato, Toshiyo; Mizutani, Kenji; Saotome, Tomonori; Noguchi, Keiichi; Kidokoro, Shun-ichi; Kuroda, Yutaka
- Abstract
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.
- Subjects
ESCHERICHIA coli; VIRAL envelope proteins; INFLUENZA; AFFINITY chromatography; VIRUS diseases
- Publication
International Journal of Molecular Sciences, 2024, Vol 25, Issue 7, p3943
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms25073943