Abstract
At the Center of Molecular Immunology (Havana, Cuba), the fusion protein SARS-CoV-2 S protein (RBD)-hFc was synthesized linking the receptor-binding domain (RBD) of the SARS-CoV-2 virus and the crystallizable fragment of a human immunoglobulin. This fusion protein was used in the construction of a diagnostic device for COVID-19 called UMELISA SARS-CoV-2-IgG. Given the relevance of this protein, the characterization of three batches (A1, A2 and A3) was carried out. The molecular weight of the protein was determined to be 120 kDa, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its isoelectric point was estimated between 8.3 and 9 by isoelectric focusing. The molecular integrity was evaluated by size exclusion liquid chromatography and SDS-PAGE after one year of the production of the protein; the presence of aggregates and fragments was detected. Batches A1 and A2 have a purity percentage higher than 95% and they can be used for the construction of new diagnostic devices.
-
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
-
Research funding: None declared.
-
Conflict of interest statement: The authors declare no conflicts of interest regarding this article.
References
1. Accinelli, R, Zhang Xu, C, Yachachin-Chavez, J, Wang, J, Caceres-Pizarro, J, Tafur-Bances, K, et al.. COVID-19: pandemic for the new virus SARS-CoV-2. Rev Peru Med Exp Salud Pública 2020;2:302–11. https://doi.org/10.17843/rpmesp.2020.372.5411.Search in Google Scholar PubMed
2. Rodriguez Martinto, J. UMELISA SARS COV 2 IgG the novel Cuban system for the diagnosis of COVID 19. Granma 2020;3:14.Search in Google Scholar
3. Tentin, S, Prendergast, F, Venyaminov, S. Accuracy of protein secondary structure from circular dichroism spectra based on immunoglobulin examples. Anal Chem 2003;321:183–7.10.1016/S0003-2697(03)00458-5Search in Google Scholar PubMed
4. He, Y, Qi, J, Xiao, L, Shen, L, Yu, W, Hu, T. Purification and characterization of the receptor-binding domain of SARS-CoV-2 spike protein from Escherichia coli. Eng Life Sci 2021;21:453–60. https://doi.org/10.1002/elsc.202000106.Search in Google Scholar PubMed PubMed Central
5. 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;581:215–25. https://doi.org/10.1038/s41586-020-2180-5.Search in Google Scholar PubMed
6. ProMab Biotechnologies Inc. Recombinant spike protein RBD (HFC TAG). California USA: Biotechnologies Inc.; 2020.Search in Google Scholar
7. Westermeier, R. Electrophoresis in practice: a guide to methods and applications of DNA and proteins separations, 5th ed. Weinheim, Germany: Wiley VCH; 2016.10.1002/9783527695188Search in Google Scholar
8. Fekete, S, Guillarme, D, Sandra, P, Sandra, K. Chromatographic, electrophoretic, and mass spectrometric methods for the analitical characterizacion of protein biofarmaceuticals. Anal Chem 2016;88:480–507. https://doi.org/10.1021/acs.analchem.5b04561.Search in Google Scholar PubMed
9. Garfin, D. Gel electroforesis of proteins. In: Essencial cell biology. Oxford: Oxford University Press; 2003:197–268 pp.10.1093/oso/9780199638314.003.0007Search in Google Scholar
10. Gene Universal. SARS-CoV-2 (2019-nCoV) spike protein (RBD) (hFc Tag). Delaware USA: GeneUniversal Inc.; 2020.Search in Google Scholar
11. Hintersteiner, B, Lingg, N, Zhang, P, Woen, S, Hoi, K, Stranner, S, et al.. Charge heterogeneity: basic antibody charge variants with increased binding to Fc receptors. MABS 2016;8:1548–60. https://doi.org/10.1080/19420862.2016.1225642.Search in Google Scholar PubMed PubMed Central
12. Smith, L, Kellener, N. Proteoform: a single term describing protein complexity. Nat Methods 2013;10:186–7. https://doi.org/10.1038/nmeth.2369.Search in Google Scholar PubMed PubMed Central
13. Mathews, C, Van Holde, K. Bioquímica, 3rd ed. Madrid: McGraw-Hill-Interamericana; 2000.Search in Google Scholar
14. Hai, Y, Harshal, C, Shengshu, H, Xi, C. One-pot three-enzyme chemoenzymatic approach to the synthesis of sialosides containing natural and non-natural functionalities. Nat Protoc 2006;1:2485–92.10.1038/nprot.2006.401Search in Google Scholar PubMed PubMed Central
15. Parr, M, Montacir, O, Montacir, H. Physicochemical characterization of biopharmaceuticals. J Pharmaceut Biomed Anal 2016;130:366–89. https://doi.org/10.1016/j.jpba.2016.05.028.Search in Google Scholar PubMed
16. Voet, D, Voet, J. Biochemistry, 4th ed. New Jersey, USA: Wiley; 2011.Search in Google Scholar
17. Kim, B. Molecular profiling, Western Blot techniques. In Methods in molecular biology. Manassas, USA: Ceres Nanosciences, Inc.; 2017, vol 1606:133–9 pp.10.1007/978-1-4939-6990-6_9Search in Google Scholar PubMed
18. Woon, D, Wook, J, Jin, C, Young, G, Soo, G, Kap, J. Detection of proteins in poliacrilamida gels using eriochrome black T rhodamina B. Anal Biochem 1998;2:118–20.10.1006/abio.1998.2813Search in Google Scholar PubMed
19. Franzini, M, Bramanti, E, Ottaviano, V, Ghiri, E, Scatena, F, Barsacchi, R, et al.. A high performance gel filtration chromatography method for gamma-glutamyltransferase fraction analysis. Anal Biochem 2008;1:374. https://doi.org/10.1016/j.ab.2007.10.025.Search in Google Scholar PubMed
20. Fekete, S, Beck, A, Veuthey, JL, Guillarme, D. Theory and practice of size exclusion chromatography for the analysis of protein aggregates. J Pharmaceut Biomed Anal 2014;101:161–73. https://doi.org/10.1016/j.jpba.2014.04.011.Search in Google Scholar PubMed
21. Agilent. Pactical guide about size exclusion chromatography for the analysis of biomolecules. USA: Agilent Technologies, Inc.; 2015.Search in Google Scholar
22. Hong, P, Koza, S, Bouvier, E. Size-exclusion chromatography for the analysis of protein biotherapeutics and their aggregates. J Liq Chromatogr Relat Technol 2012;35:2923–50. https://doi.org/10.1080/10826076.2012.743724.Search in Google Scholar PubMed PubMed Central
23. Janson, J. Protein purification: principles, high resolution methods and applications. Toronyo, Canadá: Wiley; 2011.10.1002/9780470939932Search in Google Scholar
24. Balázs, B, Fekete, J, Sipkó, E. Challenges in liquid chromatographic characterization of proteins. J Chromatogr B 2016;10:3–22.10.1016/j.jchromb.2016.04.037Search in Google Scholar PubMed
25. Xu, Y, Wang, D, Mason, B, Rossomando, T, Ning, L, Liu, D, et al.. Structure, heterogeneity and developability assessment of therapeutic antibodies. mAbs 2019;11:239–64. https://doi.org/10.1080/19420862.2018.1553476.Search in Google Scholar PubMed PubMed Central
© 2022 Walter de Gruyter GmbH, Berlin/Boston
Articles in the same Issue
- Frontmatter
- Reviews
- Anticancer properties of arylchromenes and arylchromans: an overview
- Solid state lithium ion conductors for lithium batteries
- Performance and kinetics of a fluidized bed anaerobic reactor treating distillery effluent
- Use of biochemical markers for diabetes prevention in the new decade
- Antibreast cancer activities of phytochemicals from Anonna muricata using computer-aided drug design (CADD) approach
- Alkaline-earth metal(II) complexes of salinomycin – spectral properties and antibacterial activity
- Use of heterogeneous catalysis in sustainable biofuel production
- Antibacterial, antioxidant and cytotoxic activities of the stem bark of Archidendron jiringa (Jack) I.C. Nielsen
- A review of sludge production in South Africa municipal wastewater treatment plants, analysis of handling cost and potential minimization methods
- Cu-Catalysed tandem reactions for building poly hetero atom heterocycles-green chemistry tool
- Optimizing Cr(VI) adsorption parameters on magnetite (Fe3O4) and manganese doped magnetite (MnxFe(3-x)O4) nanoparticles
- Fabaceae: a significant flavonoid source for plant and human health
- A novel application of synthesised based squarylium dyes on nylon 6, and silk woven fabrics
- Chromatographic characterization of the fusion protein SARS-CoV-2 S protein (RBD)-hFc
- Ethnopharmacology, phytochemistry and a new chemotaxonomic marker in Oldenlandia affinis (Roem. & Schult.) DC. Rubiaceae
- Extraction, isolation and characterization of secondary metabolites in the leaves of Morinda lucida from Oshiegbe in Ebonyi State
- Lead optimisation efforts on a molecular prototype of the immunomodulatory parasitic protein ES-62
- Catalytic applications of graphene oxide towards the synthesis of bioactive scaffolds through the formation of carbon–carbon and carbon–heteroatom bonds
- Synthesis, characterization, DFT and molecular docking studies of acetone O-((2,5-dichlorophenyl)sulfonyl) oxime
- Design of membrane systems
- Conceptual design and cost-efficient environmentally Benign synthesis of beta-lactams
- Synthesis and characterization of alkaloid derived hydrazones and their metal (II) complexes
- The spontaneity of chemical reactions: challenges with handling the concept and its implications
- Copper nanoparticles catalyzed carbon–heteroatom bond formation and synthesis of related heterocycles by greener procedures
- Tellurium in carbohydrate synthesis
- Conformational preferences and intramolecular hydrogen bonding patterns of tetraflavaspidic acid BBBB – a tetrameric acylphloroglucinol
- Phytochemical and antioxidant studies of Hibiscus Cannabinus seed oil
- Polyaniline mediated heterogeneous catalysis in the preparation of heterocyclic derivatives through carbon–heteroatom bond formations
- A comparison of two digestion methods and heavy metals determination in sediments
- Synthesis, properties and catalysis of quantum dots in C–C and C-heteroatom bond formations
- Recyclable magnetically retrievable nanocatalysts for C–heteroatom bond formation reactions
- Small molecules as next generation biofilm inhibitors and anti-infective agents
- Toxicity of tellurium and its compounds
- Biodegradable polycaprolactone (PCL) based polymer and composites
- Quality of poultry meat- the practical issues and knowledge based solutions
Articles in the same Issue
- Frontmatter
- Reviews
- Anticancer properties of arylchromenes and arylchromans: an overview
- Solid state lithium ion conductors for lithium batteries
- Performance and kinetics of a fluidized bed anaerobic reactor treating distillery effluent
- Use of biochemical markers for diabetes prevention in the new decade
- Antibreast cancer activities of phytochemicals from Anonna muricata using computer-aided drug design (CADD) approach
- Alkaline-earth metal(II) complexes of salinomycin – spectral properties and antibacterial activity
- Use of heterogeneous catalysis in sustainable biofuel production
- Antibacterial, antioxidant and cytotoxic activities of the stem bark of Archidendron jiringa (Jack) I.C. Nielsen
- A review of sludge production in South Africa municipal wastewater treatment plants, analysis of handling cost and potential minimization methods
- Cu-Catalysed tandem reactions for building poly hetero atom heterocycles-green chemistry tool
- Optimizing Cr(VI) adsorption parameters on magnetite (Fe3O4) and manganese doped magnetite (MnxFe(3-x)O4) nanoparticles
- Fabaceae: a significant flavonoid source for plant and human health
- A novel application of synthesised based squarylium dyes on nylon 6, and silk woven fabrics
- Chromatographic characterization of the fusion protein SARS-CoV-2 S protein (RBD)-hFc
- Ethnopharmacology, phytochemistry and a new chemotaxonomic marker in Oldenlandia affinis (Roem. & Schult.) DC. Rubiaceae
- Extraction, isolation and characterization of secondary metabolites in the leaves of Morinda lucida from Oshiegbe in Ebonyi State
- Lead optimisation efforts on a molecular prototype of the immunomodulatory parasitic protein ES-62
- Catalytic applications of graphene oxide towards the synthesis of bioactive scaffolds through the formation of carbon–carbon and carbon–heteroatom bonds
- Synthesis, characterization, DFT and molecular docking studies of acetone O-((2,5-dichlorophenyl)sulfonyl) oxime
- Design of membrane systems
- Conceptual design and cost-efficient environmentally Benign synthesis of beta-lactams
- Synthesis and characterization of alkaloid derived hydrazones and their metal (II) complexes
- The spontaneity of chemical reactions: challenges with handling the concept and its implications
- Copper nanoparticles catalyzed carbon–heteroatom bond formation and synthesis of related heterocycles by greener procedures
- Tellurium in carbohydrate synthesis
- Conformational preferences and intramolecular hydrogen bonding patterns of tetraflavaspidic acid BBBB – a tetrameric acylphloroglucinol
- Phytochemical and antioxidant studies of Hibiscus Cannabinus seed oil
- Polyaniline mediated heterogeneous catalysis in the preparation of heterocyclic derivatives through carbon–heteroatom bond formations
- A comparison of two digestion methods and heavy metals determination in sediments
- Synthesis, properties and catalysis of quantum dots in C–C and C-heteroatom bond formations
- Recyclable magnetically retrievable nanocatalysts for C–heteroatom bond formation reactions
- Small molecules as next generation biofilm inhibitors and anti-infective agents
- Toxicity of tellurium and its compounds
- Biodegradable polycaprolactone (PCL) based polymer and composites
- Quality of poultry meat- the practical issues and knowledge based solutions