Aspergillus oryzae as a host for SARS-CoV-2 RBD and NTD expression

Yıl 2024, Cilt: 33 Sayı: 2, 82 - 90

Elif Karaman Serdar Uysal

https://doi.org/10.38042/biotechstudies.1497521

Öz

The COVID-19 pandemic has increased demand for effective diagnostics, and extensive research has been conducted on the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein, which are critical for viral binding. This study focuses on the expression of NTD and RBD in pyrG auxotrophic Aspergillus oryzae for the first time. Recombinant NTD and RBD were expressed as glucoamylase-fusion proteins and purified using metal affinity chromatography. Size-exclusion chromatography was used to confirm the correct folding and purity of the recombinant proteins. Employing an enzyme-linked immunosorbent assay, the binding ability of the fusion proteins to human anti-IgG antibodies in serum samples was evaluated. The results indicated a significant and concentration-dependent interaction, affirming the functionality of the NTD and RBD fusion proteins and establishing their efficacy in antigen-antibody interactions. This study not only elucidates the usage potential of the fusion proteins in immunoassays but also addresses the suitability of the A. oryzae expression system as a biotechnological platform to produce SARS-CoV-2 proteins. Furthermore, this study lays the foundation for scalable and cost-effective mass production of effective NTD and RBD proteins in A. oryzae, opening up a new era of COVID-19 research, vaccine development, and immunoassay design.

Anahtar Kelimeler

Receptor binding domain, N-terminal domain, SARS-CoV-2, Aspergillus oryzae, Recombinant

Etik Beyan

The institutional review board approval for the study was granted by the Bezmialem Vakif University Institutional Review Board and by the Turkish Ministry of Health, and all procedures were in accordance with the approval. The subject had given informed consent for their samples to be stored and used for serological testing in the future.

Teşekkür

We thank all staff from Department of Medical Microbiology, Medical School and Beykoz Institute of Life Sciences and Biotechnology, Bezmialem Vakif University, for human serum collection.

Kaynakça

• Argentinian AntiCovid Consortium. (2020). Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells. Nature Scientific Reports, 10(1), 21779. https://doi.org/10.1038/s41598-020-78711-6
• Azar, L. M., Öncel, M. M., Karaman, E., Soysal, L. F., Fatima, A., Choi, S. B., Eyupoglu, A. E., Erman, B., Khan, A. M., & Uysal, S. (2023). Human ACE2 orthologous peptide sequences show better binding affinity to SARS-CoV-2 RBD domain: Implications for drug design. Computational and Structural Biotechnology Journal, 21, 4096-4109. https://doi.org/10.1016/j.csbj.2023.07.022
• Balasubramaniyam, A., Ryan, E., Brown, D., Hamza, T., Harrison, W., Gan, M., Sankhala, R. S., Chen, W., Martinez, E. J., Jensen, J. L., Dussupt, V., Mendez-Rivera, L., Mayer, S., King, J., Michael, N. L., Regules, J., Krebs, S., Rao, M., Matyas, G. R., Joyce, M. G., Batchelor, A. H., Gromowski, G. D., & Dutta, S. (2022). Unglycosylated soluble SARS-CoV-2 receptor binding domain (RBD) produced in E.coli combined with the army liposomal formulation containing QS21 (ALFQ) elicits neutralizing antibodies against mismatched variants. Vaccines, 11(1), 42. https://doi.org/10.3390/vaccines11010042
• Bastos, M. L., Tavaziva, G., Abidi, S. K., Campbell, J. R., Haraoui, L. P., Johnston, J. C., Lan, Z., Law, S., MacLean, E., Trajman, A., Menzies, D., Benedetti, A., & Khan, F. A. (2020). Diagnostic accuracy of serological tests for COVID-19: Systematic review and meta-analysis. BMJ, 370, 2516. https://doi.org/10.1136/bmj.m2516
• Brindha, S., & Kuroda, Y. (2022). A multi-disulfide receptor-binding domain (RBD) of the SARS-CoV-2 spike protein expressed in E. coli using a SEP-tag produces antisera interacting with the mammalian cell expressed spike (S1) protein. International Journal of Molecular Sciences, 23(3), 1703. https://doi.org/10.3390/ijms23031703
• Cao, W., Dong, C., Kim, S., Hou, D., Tai, W., Du, L., Im, W., & Zhang, X. F. (2021). Biomechanical characterization of SARS-CoV-2 spike RBD and human ACE2 protein-protein interaction. Biophysical Journal, 120, 1011–1019. https://doi.org/10.1016/j.bpj.2021.02.007
• Chen, J., Miao, L., Li, J., Li, Y., Zhu, Q., Zhou, C., Fang, H., & Chen, H. P. (2005). Receptor-binding domain of SARS-CoV spike protein: Soluble expression in E. coli, purification and functional characterization. World Journal of Gastroenterology, 11(39), 6159-6164. https://doi.org/10.3748/wjg.v11.i39.6159
• Chen, W. H., Pollet, J., Strych, U., Lee, J., Liu, Z., Kundu, R. T., Versteeg, L., Villar, M. J., Adhikari, R., Wei, J., Poveda, C., Keegan, B., Bailey, A. O., Chen, Y., Gillespie, P. M., Kimata, J. T., Zhan, B., Hotez, P.J., & Bottazzi, M. E. (2022). Yeast-expressed recombinant SARS-CoV-2 receptor binding domain RBD203-N1 as a COVID-19 protein vaccine candidate. Protein Expression and Purification, 190, 106003. https://doi.org/10.1016/j.pep.2021.106003
• Chuck, C. P., Wong, C. H., Chow, L. M., Fung, K. P., Waye, M. M., & Tsui, S. K. (2009). Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris. Virus Genes, 38(1), 1-9. https://doi.org/10.1007/s11262-008-0292-3
• Conzentino, M. S., Forchhammer, K., Souza, E. M., Pedrosa, F. O., Nogueira, M. B., Raboni, S. M., Rego, F. G. M., Zanette, D. L., Aoki, M. N., Nardin, J. M., Fornazari, B., Morales, H. M. P., Celedon, P. A. F., Lima, C. V. P., Mattar, S. B., Lin, V. H., Morello, L. G., Marchini, F. K., Reis, R. A., & Huergo, L. F. (2021). Antigen production and development of an indirect ELISA based on the nucleocapsid protein to detect human SARS-CoV-2 seroconversion. Brazilian Journal of Microbiology, 52, 2069-2073. https://doi.org/10.1007/s42770-021-00556-6
• Conzentino, M. S., Gonçalves, A. C., Paula, N. M., Rego, F. G., Zanette, D. L., Aoki, M. N., Nardin, J. M., & Huergo, L. F. (2022). A magnetic bead immunoassay to detect high affinity human IgG reactive to SARS-CoV-2 Spike S1 RBD produced in Escherichia coli. Brazilian Journal of Microbiology, 53(3), 1263-1269. https://doi.org/10.1007/s42770-022-00753-x
• Esposito, D., Mehalko, J., Drew, M., Snead, K., Wall, V., Taylor, T., Frank, P., Denson, J-P., Hong, M., Gulten, G., Sadtler, K., Messing, S., & Gillette, W. (2020). Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays. Protein Expression and Purification, 174, 105686. https://doi.org/10.1016/j.pep.2020.105686
• Fitzgerald, G. A., Komarov, A., Kaznadzey, A., Mazo, I., & Kireeva, M. L. (2021). Expression of SARS-CoV-2 surface glycoprotein fragment 319–640 in E. coli, and its refolding and purification. Protein Expression and Purification, 183, 105861. https://doi.org/10.1016/j.pep.2021.105861
• Gao, X., Peng, S., Mei, S., Liang, K., Khan, M. S. I., Vong, E. G., & Zhan, J. (2022). Expression and functional identification of recombinant SARS-CoV-2 receptor binding domain (RBD) from E. coli system. Preparative Biochemistry & Biotechnology, 52(3), 318-324. https://doi.org/10.1080/10826068.2021.1941106
• Hassanin, A. A., Raza, S. H., Ujjan, J. A., Alrashidi, A. A., Sitohy, B. M., Al-surhanee, A. A., Saad, A. M., Al-Hazani, T. M., Atallah, O. O., Al Syaad, K. M., Ahmed, A. E., Swelum, A. A., El-Saadony, M. T. & Sitohy, M. Z. (2021). Emergence, evolution, and vaccine production approaches of SARS-CoV-2 virus: Benefits of getting vaccinated and common questions. Saudi Journal of Biological Sciences, 9(4), 1981-1997. https://doi.org/10.1016/j.sjbs.2021.12.020
• He, B., Tu, Y., Jiang, C., Zhang, Z., Li, Y., & Zeng, B. (2019). Functional genomics of Aspergillus oryzae: Strategies and progress. Microorganisms, 7(103), 1-13. https://doi.org/10.3390/microorganisms7040103
• He, Y., Qi, J., Xiao, L., Shen, L., Yu, W., & Hu, T. (2021). Purification and characterization of the receptor‐binding domain of SARS‐CoV‐2 spike protein from Escherichia coli. Engineering in Life Sciences, 21(6), 453-460. https://doi.org/10.1002/elsc.202000106
• Huang, Y.; Yang, C.; Xu, X.; Xu, W., & Liu, S. (2020). Structural and functional properties of SARS-CoV-2 spike protein: Potential antivirus drug development for COVID-19. Acta Pharmacologica Sinica, 41, 1141–1149. https://doi.org/10.1038/s41401-020-0485-4
• Kalyoncu, S., Yilmaz, S., Kuyucu, A. Z., Sayili, D., Mert, O., Soyturk, H., Gullu, S., Akinturk, H., Citak, E., Arslan, M., Taskinarda, M. G., Tarman, I. O., Yilmazer Altun, G., Ozer, C., Orkut, R., Demirtas, A., Tilmensagir, I., Keles, U., Ulker, C., Aralan, G., Mercan, Y., Ozkan, M., Caglar, H. O., Arik, G., Ucar, M. C., Yildirim, M., Canavar Yildirim, T., Karadag, D., Bal, E., Erdogan, A., Senturk, S., Uzar, S., Enul, H., Adiay, C., Sarac, F., Ekiz, A. T., Abaci, I., Aksoy, O., Polat, H. U., Tekin, S., Dimitrov, S., Ozkul, A., Wingender, G., Gursel, I., Ozturk, M., & Inan, M. (2023). Process development for an effective COVID-19 vaccine candidate harboring recombinant SARS-CoV-2 delta plus receptor binding domain produced by Pichia pastoris. Nature Scientific Reports, 13(1), 5224. https://doi.org/10.1038/s41598-023-32021-9
• Karaman, E., Eyüpoğlu, A. E., Mahmoudi Azar, L., & Uysal, S. (2023). Large-scale production of anti-RNase A VHH expressed in pyrG auxotrophic Aspergillus oryzae. Current Issues Molecular Biology, 45(6), 4778-4795. https://doi.org/10.3390/cimb45060304
• Kim, W. S., Kim, J. H., Lee, J., Ka, S. Y., Chae, H. D., Jung, I., Jung, S. T., & Na, J. H. (2022). Functional expression of the recombinant spike receptor binding domain of SARS-CoV-2 Omicron in the periplasm of Escherichia coli. Bioengineering, 9(11), 670. https://doi.org/10.3390/bioengineering9110670
• Lan, J., Ge, J., Yu, J., Shan, S., Zhou, H., Fan, S., Zhang, Q., Shi, X., Wang, Q., Zhang, L., & Wang, X. (2020). Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature, 581(7807), 215-220. https://doi.org/10.1038/s41586-020-2180-5
• Li, W., Moore, M. J., Vasilieva, N. J., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C. Choe, H., & Farzan, M. (2003). Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature, 426, 450–454. https://doi.org/10.1038/nature02145
• Liu, B., Yin, Y., Liu, Y., Wang, T., Sun, P., Ou, Y., Gong, X., Hou, X., Zhang, J., Ren, H., Luo, S., Ke, Q., Yao, Y., Xu, J., & Wu, J. (2022). A vaccine based on the receptor-binding domain of the spike protein expressed in glycoengineered Pichia pastoris targeting SARS-CoV-2 stimulates neutralizing and protective antibody responses. Engineering, 13, 107-115. https://doi.org/10.1016/j.eng.2021.06.012
• Maffei, M., Montemiglio, L. C., Vitagliano, G., Fedele, L., Sellathurai, S., Bucci, F., Compagnone, M., Chiarini, V., Exertier, C., Muzi, A., Roscilli, G., Vallone, B., & Marra, E. (2021). The nuts and bolts of SARS-CoV-2 spike receptor-binding domain heterologous expression. Biomolecules, 11(12), 1812. https://doi.org/10.3390/biom11121812
• Márquez-Ipiña, A. R., González-González, E., Rodríguez-Sánchez, I. P., Lara-Mayorga, I. M., Mejía-Manzano, L. A., Sánchez-Salazar, M. G., González-Valdez, J. G., Ortiz-Lopez, R., Rojas-Martinez, A., Santiago, G. T., & Alvarez, M. M. (2021). Serological test to determine exposure to SARS-CoV-2: ELISA based on the receptor-binding domain of the spike protein (S-RBDN318-V510) expressed in Escherichia coli. Diagnostics, 11(2), 271. https://doi.org/10.3390/diagnostics11020271
• McAndrews, K. M., Dowlatshahi, D. P., Dai, J., Becker, L. M., Hensel, J., Snowden, L. M., Leveille, J. M., Brunner, M. R., Holden, K. W., Hopkins, N. S., Harris, A. M., Kumpati, J., Whitt, M. A., Lee, J. J., Ostrosky-Zeichner, L. L., Papanna, R., LeBleu, V. S., Allison, J. P., & Kalluri, R. (2020). Heterogeneous antibodies against SARS-CoV-2 spike receptor binding domain and nucleocapsid with implications for COVID-19 immunity. JCI Insight, 5(18). https://doi.org/10.1172/jci.insight.142386
• McGuire, B. E., Mela, J. E., Thompson, V. C., Cucksey, L. R., Stevens, C. E., McWhinnie, R. L., Winkler, D. F. H., Pelech, S., & Nano, F. E. (2022). Escherichia coli recombinant expression of SARS-CoV-2 protein fragments. Microbial Cell Factories, 21(1), 1-13. https://doi.org/10.1186/s12934-022-01753-0
• Merkuleva, I. A., Shcherbakov, D. N., Borgoyakova, M. B., Shanshin, D. V., Rudometov, A. P., Karpenko, L. I., Belenkaya, S. V., Isaeva, A. A., Nesmeyanova, V. S., Kazachinskaia, E. I., Volosnikova, E. A., Esina, T. I., Zaykovskaya, A. V., Pyankov, O. V., Borisevich, S. S., Shelemba, A. A., Chikaev, A. N., & Ilyichev, A. A. (2022). Comparative immunogenicity of the recombinant receptor-binding domain of protein S SARS-CoV-2 obtained in prokaryotic and mammalian expression systems. Vaccines, 10(1), 96. https://doi.org/10.3390/vaccines10010096
• Mi, T., Wang, T., Xu, H., Sun, P., Hou, X., Zhang, X., Ke, Q., Liu, J., Hu, S., Wu, J., & Liu, B. (2022). Kappa-RBD produced by glycoengineered Pichia pastoris elicited high neutralizing antibody titers against pseudoviruses of SARS-CoV-2 variants. Virology, 569, 56-63. https://doi.org/10.1016/j.virol.2022.03.001
• Ntana, F., Mortensen, U.H., Sarazin, C., & Figge, R. (2020). Aspergillus: A powerful protein production platform. Catalysts, 10(9), 1064; https://doi.org/10.3390/catal10091064
• Pino, M., Abid, T., Pereira Ribeiro, S., Edara, V. V., Floyd, K., Smith, J. C., Latif, M. B., Pacheco-Sanchez, G., Dutta, D., Wang, S., Gumber, S., Kirejczyk, S., Cohen, J., Stammen, R. L., Jean, S. M., Wood, J. S., Connor-Stroud, F., Pollet, J., Chen, W., Wei, J., Zhan, B., Lee, J., Liu, Z., Strych, U., Shenvi, N., Easley, K., Weiskopf, D., Sette, A., Pollara, J., Mielke, D., Gao, H., Eisel, N., Lebranche, C. C., Shen, X., Ferrari, G., Tomaras, G. D., Montefiori, D. C., Sekaly, R. P., Vanderford, T. H., Tomai, M. A., Fox, c. B., Suthar, M. S., Kozlowski, P. A., Hotez, P. J., Paiardini, M., Bottazzi, M. E., & Kasturi, S. P. (2021). A yeast-expressed RBD-based SARS-CoV-2 vaccine formulated with 3M-052-alum adjuvant promotes protective efficacy in non-human primates. Science Immunology, 6(61), eabh3634. https://doi.org/10.1126/sciimmunol.abh3634
• Prahlad, J., Struble, L. R., Lutz, W. E., Wallin, S. A., Khurana, S., Schnaubelt, A., Broadhurst, M. J., Bayles, K. W., & Borgstahl, G. E. (2021). CyDisCo production of functional recombinant SARS‐CoV‐2 spike receptor binding domain. Protein Science, 30(9), 1983-1990. https://doi.org/10.1002/pro.4152
• Sakai, K., Kinoshita, H., & Nihira, T. (2012). Heterologous expression system in Aspergillus oryzae for fungal biosynthetic gene clusters of secondary metabolites. Applied Microbiology and Biotechnology, 93(5), 2011-2022. https://doi.org/10.1007/s00253-011-3657-9
• Shang, J., Ye, G., Shi, K., Wan, Y., Luo, C., Aihara, H., Geng, Q., Auerbach, A., & Fang, L. (2020). Structural basis of receptor recognition by SARS-CoV-2. Nature, 581(7807), 221-224. https://doi.org/10.1038/s41586-020-2179-y
• Tantiwiwat, T., Thaiprayoon, A., Siriatcharanon, A. K., Tachaapaikoon, C., Plongthongkum, N., & Waraho-Zhmayev, D. (2023). Utilization of receptor-binding domain of SARS-CoV-2 spike protein expressed in Escherichia coli for the development of neutralizing antibody assay. Molecular Biotechnology, 65(4), 598-611. https://doi.org/10.1007/s12033-022-00563-4
• Tay, M. Z., Poh, C. M., Rénia, L., MacAry , P. A., & Ng, L. F. (2020). The trinity of COVID-19: immunity, inflammation and intervention. Nature Reviews Immunology, 20(6), 363-374. https://doi.org/10.1038/s41577-020-0311-8
• Tozetto-Mendoza, T. R., Kanunfre, K. A., Vilas-Boas, L. S., Espinoza, E. P. S., Paião, H. G. O., Rocha, M. C., de Paula, A. V., de Oliveira, M., S., Zampelli, D. B., Vieira Jr., J. M., Buss, L., Costa, S., F., Sabino, E. C., Witkin, S. S., Okay, T. S., & Mendes-Correa, M. C. (2021). Nucleoprotein-based ELISA for detection of SARS-COV-2 IgG antibodies: Could an old assay be suitable for serodiagnosis of the new coronavirus?. Journal of Virological Methods, 290, 114064. https://doi.org/10.1016/j.jviromet.2021.114064
• Tripathi, N.K. (2016). Production and purification of recombinant proteins from Escherichia coli. Chem Bio Eng Reviews, 3, 116–133. https://doi.org/10.1002/cben.201600002
• Walls, A. C., Park, Y. J., Tortorici, M. A., Wall, A., McGuire, A. T., & Veesler, D. (2020). Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell, 181(2), 281-292. https://doi.org/10.1016/j.cell.2020.02.058
• Wrapp, D., Wang, N., Corbett, K.S., Goldsmith, J.A., Hsieh, C., Abiona, O., Graham, B.S., & McLellan, J.S. (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science, 367, 1260–1263. https://doi.org/10.1126/science.abb2507
• Xing, H., Zhu, L., Wang, P., Zhao, G., Zhou, Z., Yang, Y., Zou, H., & Yan, X. (2022). Display of receptor-binding domain of SARS-CoV-2 spike protein variants on the Saccharomyces cerevisiae cell surface. Frontiers in Immunology, 13, 935573. https://doi.org/10.3389/fimmu.2022.935573
• Zang, J., Zhu, Y., Zhou, Y., Gu, C., Yi, Y., Wang, S., Xu, S., Hu, G., Du, S., Yin, Y., Wang, Y., Yang, Y., Zhang, X., Wang, H., Yin, F., Zhang, C., Deng, Q., Xie, Y., & Huang, Z. (2021). Yeast-produced RBD-based recombinant protein vaccines elicit broadly neutralizing antibodies and durable protective immunity against SARS-CoV-2 infection. Cell Discovery, 7(1), 71. https://doi.org/10.1038/s41421-021-00315-9
• Zhu, G., Zhu, C., Zhu, Y., & Sun, F. (2020). Minireview of progress in the structural study of SARS-CoV-2 proteins. Current Research in Microbial Sciences, 1, 53-61. https://doi.org/10.1016/j.crmicr.2020.06.003