Expression of Receptor Binding Domain (RBD) from Coronavirus Spike Protein Fused to Carboxylic Terminal of Clostridium perfringens Enterotoxin (c-CPE) in Pichia pastoris

How to Cite:Behvandi E, Bagherpour G, Nedaei K, Kaboli S, Johari B, Expression of Receptor Binding Domain (RBD) from Coronavirus Spike Protein Fused to Carboxylic Terminal of Clostridium perfringens Enterotoxin (c-CPE) in Pichia pastoris.koomesh.2024;26(2):e149316.https://doi.org/10.69107/koomesh-149316.

Abstract

Background: To enhance mucosal IgA, administering intra-nasal antigen transfer may be considered. One challenge with this approach is antigenic factors' limited penetration of nasal mucosa. This research utilized c-CPE, the carboxylic end-section of the enterotoxin Clostridium perfringens. Specifically, the RBD-c-CPE fusion structure was cloned in a bacterial host and integrated into the yeast host genome for protein expression.

Objectives: The primary aim of this investigation is to develop methods: Following the construction of the expression structure, it was then cloned into the yeast vector a coronavirus vaccine that can elicit sufficient immunity in the host.

Methods: Materials pPICZαA: Subsequently, the TOP10F bacterium was transformed with the recombinant plasmid, and plasmid extraction was carried out. This was followed by the transformation of yeast strain X33 with linearized recombinant plasmids. Positive clones obtained from the overnight sowing process were utilized for the expression stages. To confirm protein secretion in the yeast medium, the protein concentration, western blot, and ECL were performed.

Results: Agarose gel electrophoresis results indicated the successful construction of the RBD-c-CPE gene. Colony PCR analysis of both TOP10F bacteria and Pichia pastoris yeast post-transformation confirmed the effective cloning of the recombinant pPIC-RC plasmid in TOP10F bacteria and the successful integration of the recombinant plasmid into the yeast genome, respectively. Moreover, Western blot analysis provided conclusive evidence of the successful expression of the RBD-c-CPE fusion protein in Pichia pastoris yeast.

Conclusions: The RBD-c-CPE has been effectively integrated and produced within the yeast host genome following its cloning in a bacterial host. Further enhancing the fused protein's expression can serve as a potential candidate for preclinical trials on animals for a COVID-19 mucosal vaccine.

Keywords

Recombinant Fusion Proteins

Cloning

Spike Protein

Pichia pastoris

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References

1.
Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579(7798):270-3. [PubMed ID:32015507]. [PubMed Central ID:PMC7095418]. https://doi.org/10.1038/s41586-020-2012-7.
2.
Yan R, Zhang Y, Li Y, Xia L, Guo Y, Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science. 2020;367(6485):1444-8. [PubMed ID:32132184]. [PubMed Central ID:PMC7164635]. https://doi.org/10.1126/science.abb2762.
3.
Peng Y, Du N, Lei Y, Dorje S, Qi J, Luo T, et al. Structures of the SARS-CoV-2 nucleocapsid and their perspectives for drug design. EMBO J. 2020;39(20):e105938. [PubMed ID:32914439]. [PubMed Central ID:PMC7560215]. https://doi.org/10.15252/embj.2020105938.
4.
Mandala VS, McKay MJ, Shcherbakov AA, Dregni AJ, Kolocouris A, Hong M. Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers. Nat Struct Mol Biol. 2020;27(12):1202-8. [PubMed ID:33177698]. [PubMed Central ID:PMC7718435]. https://doi.org/10.1038/s41594-020-00536-8.
5.
Siu YL, Teoh KT, Lo J, Chan CM, Kien F, Escriou N, et al. The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles. J Virol. 2008;82(22):11318-30. [PubMed ID:18753196]. [PubMed Central ID:PMC2573274]. https://doi.org/10.1128/JVI.01052-08.
6.
Huang WC, Zhou S, He X, Chiem K, Mabrouk MT, Nissly RH, et al. SARS-CoV-2 RBD Neutralizing Antibody Induction is Enhanced by Particulate Vaccination. Adv Mater. 2020;32(50):e2005637. [PubMed ID:33111375]. [PubMed Central ID:PMC7645956]. https://doi.org/10.1002/adma.202005637.
7.
Lan J, Ge J, Yu J, Shan S, Zhou H, Fan S, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 2020;581(7807):215-20. [PubMed ID:32225176]. https://doi.org/10.1038/s41586-020-2180-5.
8.
Wang X, Tu W. A promising vaccine candidate against COVID-19. Mol Biomed. 2020;1(1):8. [PubMed ID:34765993]. [PubMed Central ID:PMC7525074]. https://doi.org/10.1186/s43556-020-00008-x.
9.
Morin PJ. Claudin proteins in human cancer: promising new targets for diagnosis and therapy. Cancer Res. 2005;65(21):9603-6. [PubMed ID:16266975]. https://doi.org/10.1158/0008-5472.CAN-05-2782.
10.
Robertson SL, Smedley JG, 3rd, Singh U, Chakrabarti G, Van Itallie CM, Anderson JM, et al. Compositional and stoichiometric analysis of Clostridium perfringens enterotoxin complexes in Caco-2 cells and claudin 4 fibroblast transfectants. Cell Microbiol. 2007;9(11):2734-55. [PubMed ID:17587331]. https://doi.org/10.1111/j.1462-5822.2007.00994.x.
11.
Katahira J, Inoue N, Horiguchi Y, Matsuda M, Sugimoto N. Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin. J Cell Biol. 1997;136(6):1239-47. [PubMed ID:9087440]. [PubMed Central ID:PMC2132509]. https://doi.org/10.1083/jcb.136.6.1239.
12.
Bagherpour G, Ghasemi H, Zand B, Zarei N, Roohvand F, Ardakani EM, et al. Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice. Front Microbiol. 2018;9:723. [PubMed ID:29706942]. [PubMed Central ID:PMC5908956]. https://doi.org/10.3389/fmicb.2018.00723.
13.
Rahbar Z, Nazarian S, Dorostkar R, Sotoodehnejadnematalahi F, Amani J. Recombinant expression of SARS-CoV-2 receptor binding domain (RBD) in Escherichia coli and its immunogenicity in mice. Iran J Basic Med Sci. 2022;25(9):1110-6. [PubMed ID:36246069]. [PubMed Central ID:PMC9526882]. https://doi.org/10.22038/IJBMS.2022.65045.14333.
14.
Choque-Guevara R, Poma-Acevedo A, Montesinos-Millan R, Rios-Matos D, Gutierrez-Manchay K, Montalvan-Avalos A, et al. Squalene in oil-based adjuvant improves the immunogenicity of SARS-CoV-2 RBD and confirms safety in animal models. PLoS One. 2022;17(8):e0269823. [PubMed ID:35998134]. [PubMed Central ID:PMC9397949]. https://doi.org/10.1371/journal.pone.0269823.
15.
Merkuleva IA, Shcherbakov DN, Borgoyakova MB, Shanshin DV, Rudometov AP, Karpenko LI, et al. Comparative Immunogenicity of the Recombinant Receptor-Binding Domain of Protein S SARS-CoV-2 Obtained in Prokaryotic and Mammalian Expression Systems. Vaccines (Basel). 2022;10(1). [PubMed ID:35062757]. [PubMed Central ID:PMC8779843]. https://doi.org/10.3390/vaccines10010096.
16.
Khorattanakulchai N, Srisutthisamphan K, Shanmugaraj B, Manopwisedjaroen S, Rattanapisit K, Panapitakkul C, et al. A recombinant subunit vaccine candidate produced in plants elicits neutralizing antibodies against SARS-CoV-2 variants in macaques. Front Plant Sci. 2022;13:901978. [PubMed ID:36247553]. [PubMed Central ID:PMC9555276]. https://doi.org/10.3389/fpls.2022.901978.
17.
Limonta-Fernandez M, Chinea-Santiago G, Martin-Dunn AM, Gonzalez-Roche D, Bequet-Romero M, Marquez-Perera G, et al. An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen. N Biotechnol. 2022;72:11-21. [PubMed ID:35953030]. [PubMed Central ID:PMC9359770]. https://doi.org/10.1016/j.nbt.2022.08.002.
18.
Yang S, Li Y, Dai L, Wang J, He P, Li C, et al. Safety and immunogenicity of a recombinant tandem-repeat dimeric RBD-based protein subunit vaccine (ZF2001) against COVID-19 in adults: two randomised, double-blind, placebo-controlled, phase 1 and 2 trials. Lancet Infect Dis. 2021;21(8):1107-.
19.
[PubMed ID:33773111]. [PubMed Central ID:PMC7990482]. https://doi.org/10.1016/S1473-3099(21)00127-4.
20.
Min L, Sun Q. Antibodies and Vaccines Target RBD of SARS-CoV-2. Front Mol Biosci. 2021;8:671633. [PubMed ID:33968996]. [PubMed Central ID:PMC8100443]. https://doi.org/10.3389/fmolb.2021.671633.
21.
Vartak A, Sucheck SJ. Recent Advances in Subunit Vaccine Carriers. Vaccines (Basel). 2016;4(2). [PubMed ID:27104575]. [PubMed Central ID:PMC4931629]. https://doi.org/10.3390/vaccines4020012.
22.
Premkumar L, Segovia-Chumbez B, Jadi R, Martinez DR, Raut R, Markmann A, et al. The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients. Sci Immunol. 2020;5(48). [PubMed ID:32527802]. [PubMed Central ID:PMC7292505]. https://doi.org/10.1126/sciimmunol.abc8413.
23.
Morrow Jr KJ. Improving Protein Production Processes. 2007. Available from: https://www.genengnews.com/insights/improving-protein-production-processes/.
24.
Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA. Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol. 1989;9(3):1316-23. [PubMed ID:2657390]. [PubMed Central ID:PMC362724]. https://doi.org/10.1128/mcb.9.3.1316-1323.1989.
25.
Higgins DR. Overview of protein expression in Pichia pastoris. Curr Protoc Protein Sci. 1995;Chapter 5:Unit5 7. [PubMed ID:18429188]. https://doi.org/10.1002/0471140864.ps0507s02.
26.
Argentinian AntiCovid Consortium. Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells. Sci Rep. 2020;10(1):21779. [PubMed ID:33311634]. [PubMed Central ID:PMC7732851]. https://doi.org/10.1038/s41598-020-78711-6.
27.
Hou XC, Xu HF, Liu Y, Sun P, Ding LW, Yue JJ, et al. A Vaccine with Multiple Receptor-Binding Domain Subunit Mutations Induces Broad-Spectrum Immune Response against SARS-CoV-2 Variants of Concern. Vaccines (Basel). 2022;10(10). [PubMed ID:36298518]. [PubMed Central ID:PMC9609383]. https://doi.org/10.3390/vaccines10101653.
28.
Chen WH, Pollet J, Strych U, Lee J, Liu Z, Kundu RT, et al. Yeast-expressed recombinant SARS-CoV-2 receptor binding domain RBD203-N1 as a COVID-19 protein vaccine candidate. Protein Expr Purif. 2022;190:106003. [PubMed ID:34688919]. [PubMed Central ID:PMC8529586]. https://doi.org/10.1016/j.pep.2021.106003.
29.
Gattinger P, Kratzer B, Tulaeva I, Niespodziana K, Ohradanova-Repic A, Gebetsberger L, et al. Vaccine based on folded receptor binding domain-PreS fusion protein with potential to induce sterilizing immunity to SARS-CoV-2 variants. Allergy. 2022;77(8):2431-45. [PubMed ID:35357709]. [PubMed Central ID:PMC9111473]. https://doi.org/10.1111/all.15305.
30.
Limonta-Fernández M, Chinea-Santiago G, Martín-Dunn AM, Gonzalez-Roche D, Bequet-Romero M, Marquez-Perera G, et al. An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen. New Biotechnol. 2021;72:11-21. https://doi.org/10.1016/j.nbt.2022.08.002.
31.
Lee NP, Cheng CY. Nitric oxide/nitric oxide synthase, spermatogenesis, and tight junction dynamics. Biol Reprod. 2004;70(2):267-76. [PubMed ID:14522829]. https://doi.org/10.1095/biolreprod.103.021329.
32.
Piontek A, Eichner M, Zwanziger D, Beier LS, Protze J, Walther W, et al. Targeting claudin-overexpressing thyroid and lung cancer by modified Clostridium perfringens enterotoxin. Mol Oncol. 2020;14(2):261-76. [PubMed ID:31825142]. [PubMed Central ID:PMC6998413]. https://doi.org/10.1002/1878-0261.12615.
33.
Pahle J, Kobelt D, Aumann J, Behrens D, Daberkow O, Mokritzkij M, et al. Effective Oncoleaking Treatment of Pancreatic Cancer by Claudin-Targeted Suicide Gene Therapy with Clostridium perfringens Enterotoxin (CPE). Cancers (Basel). 2021;13(17). [PubMed ID:34503203]. [PubMed Central ID:PMC8431234]. https://doi.org/10.3390/cancers13174393.
34.
Zuercher AW, Coffin SE, Thurnheer MC, Fundova P, Cebra JJ. Nasal-associated lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular immune responses. J Immunol. 2002;168(4):1796-803. [PubMed ID:11823512]. https://doi.org/10.4049/jimmunol.168.4.1796.
35.
Kakutani H, Kondoh M, Fukasaka M, Suzuki H, Hamakubo T, Yagi K. Mucosal vaccination using claudin-4-targeting. Biomaterials. 2010;31(20):5463-71. [PubMed ID:20398936]. https://doi.org/10.1016/j.biomaterials.2010.03.047.
36.
Kemble G, Greenberg H. Novel generations of influenza vaccines. Vaccine. 2003;21(16):1789-95. [PubMed ID:12686096]. https://doi.org/10.1016/s0264-410x(03)00074-4.
37.
Suzuki H, Watari A, Hashimoto E, Yonemitsu M, Kiyono H, Yagi K, et al. C-Terminal Clostridium perfringens Enterotoxin-Mediated Antigen Delivery for Nasal Pneumococcal Vaccine. PLoS One. 2015;10(5):e0126352. [PubMed ID:26018248]. [PubMed Central ID:PMC4446347]. https://doi.org/10.1371/journal.pone.0126352.
38.
Souod N, Kargar M, Hoseini MH, Jafarinia M. Fusion-expressed CtxB-TcpA-C-CPE improves both systemic and mucosal humoral and T-cell responses against cholera in mice. Microb Pathog. 2021;157:104978. [PubMed ID:34022352]. https://doi.org/10.1016/j.micpath.2021.104978.
39.
Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013;65(10):1357-69. [PubMed ID:23026637]. [PubMed Central ID:PMC3726540]. https://doi.org/10.1016/j.addr.2012.09.039.
40.
Suzuki H, Kakutani H, Kondoh M, Watari A, Yagi K. The safety of a mucosal vaccine using the C-terminal fragment of Clostridium perfringens enterotoxin. Pharmazie. 2010;65(10):766-9. [PubMed ID:21105580].

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Author(s):

Elham Behvandi:[PubMed][Scholar]
Ghasem Bagherpour:[PubMed][Scholar]
Keivan Nedaei:[PubMed][Scholar]
Saeid Kaboli:[PubMed][Scholar]
Behrooz Johari:[PubMed][Scholar]