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: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-04-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/25/7/3943 |
| _version_ | 1797212487800586240 |
|---|---|
| author | Thao Tu Tharangani Rathnayaka Toshiyo Kato Kenji Mizutani Tomonori Saotome Keiichi Noguchi Shun-ichi Kidokoro Yutaka Kuroda |
| author_facet | Thao Tu Tharangani Rathnayaka Toshiyo Kato Kenji Mizutani Tomonori Saotome Keiichi Noguchi Shun-ichi Kidokoro Yutaka Kuroda |
| author_sort | Thao Tu |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-24T10:43:10Z |
| format | Article |
| id | doaj.art-3d6fdab8b22c4140a27227cd9a1d5c54 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-04-24T10:43:10Z |
| publishDate | 2024-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-3d6fdab8b22c4140a27227cd9a1d5c542024-04-12T13:20:11ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672024-04-01257394310.3390/ijms25073943Design and <i>Escherichia coli</i> Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD)Thao Tu0Tharangani Rathnayaka1Toshiyo Kato2Kenji Mizutani3Tomonori Saotome4Keiichi Noguchi5Shun-ichi Kidokoro6Yutaka Kuroda7Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi 184-8588, Tokyo, JapanDepartment of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi 184-8588, Tokyo, JapanNMR Group, Smart-Core-Facility Promotion Organization, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi 184-8588, Tokyo, JapanGraduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro, Yokohama 230-0045, Kanagawa, JapanDepartment of Materials Science and Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka-cho, Nagaoka-shi 940-2188, Niigata, JapanNMR Group, Smart-Core-Facility Promotion Organization, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi 184-8588, Tokyo, JapanDepartment of Materials Science and Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka-cho, Nagaoka-shi 940-2188, Niigata, JapanDepartment of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi 184-8588, Tokyo, JapanRefolding 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.https://www.mdpi.com/1422-0067/25/7/3943influenza AH1N1receptor-binding domainrefoldingNMR<i>E. coli</i> expression system |
| spellingShingle | Thao Tu Tharangani Rathnayaka Toshiyo Kato Kenji Mizutani Tomonori Saotome Keiichi Noguchi Shun-ichi Kidokoro Yutaka Kuroda Design and <i>Escherichia coli</i> Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD) International Journal of Molecular Sciences influenza A H1N1 receptor-binding domain refolding NMR <i>E. coli</i> expression system |
| title | Design and <i>Escherichia coli</i> Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD) |
| title_full | Design and <i>Escherichia coli</i> Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD) |
| title_fullStr | Design and <i>Escherichia coli</i> Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD) |
| title_full_unstemmed | Design and <i>Escherichia coli</i> Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD) |
| title_short | Design and <i>Escherichia coli</i> Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD) |
| title_sort | design and i escherichia coli i expression of a natively folded multi disulfide bonded influenza h1n1 pr8 receptor binding domain rbd |
| topic | influenza A H1N1 receptor-binding domain refolding NMR <i>E. coli</i> expression system |
| url | https://www.mdpi.com/1422-0067/25/7/3943 |
| work_keys_str_mv | AT thaotu designandiescherichiacoliiexpressionofanativelyfoldedmultidisulfidebondedinfluenzah1n1pr8receptorbindingdomainrbd AT tharanganirathnayaka designandiescherichiacoliiexpressionofanativelyfoldedmultidisulfidebondedinfluenzah1n1pr8receptorbindingdomainrbd AT toshiyokato designandiescherichiacoliiexpressionofanativelyfoldedmultidisulfidebondedinfluenzah1n1pr8receptorbindingdomainrbd AT kenjimizutani designandiescherichiacoliiexpressionofanativelyfoldedmultidisulfidebondedinfluenzah1n1pr8receptorbindingdomainrbd AT tomonorisaotome designandiescherichiacoliiexpressionofanativelyfoldedmultidisulfidebondedinfluenzah1n1pr8receptorbindingdomainrbd AT keiichinoguchi designandiescherichiacoliiexpressionofanativelyfoldedmultidisulfidebondedinfluenzah1n1pr8receptorbindingdomainrbd AT shunichikidokoro designandiescherichiacoliiexpressionofanativelyfoldedmultidisulfidebondedinfluenzah1n1pr8receptorbindingdomainrbd AT yutakakuroda designandiescherichiacoliiexpressionofanativelyfoldedmultidisulfidebondedinfluenzah1n1pr8receptorbindingdomainrbd |