Design and <i>Escherichia coli</i> Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD)
Refolding multi-disulfide bonded proteins expressed in <i>E. coli</i> into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the <i>E. coli</i> expression of viral envelope proteins, such as the RBD (Receptor-B...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-04-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/25/7/3943 |
| Summary: | Refolding multi-disulfide bonded proteins expressed in <i>E. coli</i> into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the <i>E. coli</i> 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 <i>E. coli</i> 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 <sup>15</sup>N-HSQC NMR spectrum exhibited several <sup>1</sup>H-<sup>15</sup>N 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 <i>E. coli</i> expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection. |
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| ISSN: | 1661-6596 1422-0067 |