Ukr.Biochem.J. 2021; Volume 93, Issue 6, Nov-Dec, pp. 31-45


Production of recombinant SARS-COV-2 proteins and diphtheria toxoid CRM197-based fusion

O. I. Krynina1, S. I. Romaniuk1, O. B. Gorbatiuk1,2,
O. H. Korchynskyi1,3,4, А. V. Rebriiev1, Ya. S. Kulyk1,
Ye. O. Kozadaieva1, A. A. Siromolot1,5, M. M. Guzyk1,
D. V. Kolybo1*, S. V. Komisarenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2State Institute of Genetic and Regenerative Medicine, National Academy of Medical Sciences of Ukraine, Kyiv;
3Centre for Innovative Research in Medical and Natural Sciences, Faculty of Medicine, University of Rzeszow, Rzeszow, Poland;
4S. Gzhytskyi National University of Veterinary Medicine and Biotechnologies, Lviv, Ukraine;
5ESC “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv, Ukraine;

Received: 10 October 2021; Accepted: 12 November 2021

The quickly emerged global COVID-19 pandemic raised a desperate need in the development of protecting vaccines targeting this disease. Therefore, a generation of effective producers of recombinant SARS-CoV-2 proteins became an urgent task. Its resolving contributes to the study of functional SARS-CoV-2 properties, as well as will allow developing the domestic COVID-19 vaccine in Ukraine, thus playing an important strategic role in tackling the pandemics. The aim of the study was to generate prokaryotic and eukaryotic producers of recombinant SARS-CoV-2 proteins and to isolate nucleocapsid (N) protein, receptor-binding domain (RBD) of spike (S) protein, as well as RBD fused to the carrier – diphtheria toxoid CRM197. For this purpose, appropriate genetic constructs, in particular, replication deficient recombinant AdvC5-based adenoviral vectors expressing the SARS-CoV-2 proteins and CRM197-fused conjugate were created through methods of molecular biology and genetic engineering. Restriction analysis and/or DNA sequencing confirmed that we created the correct constructs. Immobilized metal affinity chromatography was used to purify the recombinant proteins. Compliance of their properties was confirmed by the results from polyacrylamide gel electrophoresis, Western blotting, immunoenzymatic assay and MALDI-TOF mass spectrometry. As a result, we generated E. coli Rosetta (DE3) bacterial strain and HEK293 cell line producing recombinant SARS-CoV-2 proteins and CRM197-based fusion. In addition, pure N protein, RBD of S protein and RBD-CRM197 fusion protein were isolated. The obtained recombinant SARS-CoV-2 proteins can be used to study immunogenic and antigenic properties of the SARS-CoV-2 proteins. Cells producing recombinant SARS-CoV-2 proteins and RBD-CRM197 fusion protein are able to provide cheap and safe synthesis of the antigenic substances for domestic development and production of immunodiagnostics for COVID-19 and COVID-19 vaccines in Ukraine.

Keywords: , , , , , ,


  1. Komisarenko S. The global coronavirus crisis. K.: LAT&K, 2020. 120 p. (In Ukrainian).
  2. COVID-19 dashboard by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University (JHU). Available at (accessed, November 7, 2021).
  3. World Health Organization. COVID-19 vaccine tracker and landscape. Available at (accessed, November 5, 2021).
  4. PFIZER and BIONTECH achieve first authorization in the world for a vaccine to combat COVID19. (accessed, December 2, 2020).
  5. Zimmer C, Corum J, Wee S-L. Coronavirus vaccine tracker. Available at (accessed, November 4, 2021).
  6. Corum J, Zimmer C. How the Novavax vaccine works. Available at (accessed, May 7, 2021).
  7. Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, Graham BS, McLellan JS. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;367(6483):1260-1263. PubMed, PubMedCentralCrossRef
  8. Ahlén G, Frelin L, Nikouyan N, Weber F, Höglund U, Larsson O, Westman M, Tuvesson O, Gidlund EK, Cadossi M, Appelberg S, Mirazimi A, Sällberg M. The SARS-CoV-2 N Protein Is a Good Component in a Vaccine. J Virol. 2020;94(18):e01279-20. PubMed, PubMedCentralCrossRef
  9. Malito E , Bursulaya B, Chen C, Lo Surdo P, Picchianti M, Balducci E, Biancucci M, Brock A, Berti F, Bottomley MJ, Nissum M, Costantino P, Rappuoli R, Spraggon G. Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197. Proc Natl Acad Sci USA. 2012;109(14):5229-5234. PubMed, PubMedCentralCrossRef
  10. Bröker M, Berti F, Schneider J, Vojtek I. Polysaccharide conjugate vaccine protein carriers as a “neglected valency” – Potential and limitations. Vaccine. 2017;35(25):3286-3294. PubMed, CrossRef
  11. Jaffe J, Wucherer K, Sperry J, Zou Q, Chang Q, Massa MA, Bhattacharya K, Kumar S, Caparon M, Stead D, Wright P, Dirksen A, Francis MB. Effects of Conformational Changes in Peptide-CRM 197 Conjugate Vaccines. Bioconjug Chem. 2019;30(1):47-53. PubMedCrossRef
  12. Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979;7(6):1513-1523. PubMed, PubMedCentralCrossRef
  13. Labyntsev AJ, Korotkevych NV, Manoilov KJ, Kaberniuk AA, Kolybo DV, Komisarenko SV. Recombinant fluorescent models for studying the diphtheria toxin. Russ J Bioorganic Chem. 2014;40(4):401-409. PubMed, CrossRef
  14. Heckman KL, Pease LR. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc. 2007;2(4):924-932. PubMed, CrossRef
  15. Rudolph R, Lilie H. In vitro folding of inclusion body proteins. FASEB J. 1996;10(1):49-56. PubMed
  16. Basu A, Li X, Leong SSJ. Refolding of proteins from inclusion bodies: rational design and recipes. Appl Microbiol Biotechnol. 2011;92(2):241-251. PubMedCrossRef
  17. Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987;166(2):368-379. PubMed, CrossRef
  18. Expasy Swiss Bioinformatics Resource Portal. (accessed, November 5, 2021).
  19. Protein Data Bank. (accessed, November 5, 2021).
  20. Quitterer U, Pohl A, Langer A, Koller S, Abdalla S. A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor. Biochem Biophys Res Commun. 20110;409(3):544-549. PubMed, CrossRef
  21. Yu K, Liu C, Kim BG, Lee DY. Synthetic fusion protein design and applications. Biotechnol Adv. 2015;33(1):155-164. PubMed, CrossRef
  22. Grant OC, Montgomery D, Ito K, Woods RJ. Analysis of the SARS-CoV-2 spike protein glycan shield reveals implications for immune recognition. Sci Rep. 2020;10(1):14991. PubMed, PubMedCentralCrossRef
  23. Supekar NT, Shajahan A, Gleinich AS, Rouhani DS, Heiss C, Chapla DG, Moremen KW, Azadi P. Variable posttranslational modifications of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein. Glycobiology. 2021;31(9):1080-1092. PubMed, PubMedCentralCrossRef
  24. Zhao Q, Gao Y, Xiao M, Huang X, Wu X. Synthesis and immunological evaluation of synthetic peptide based anti-SARS-CoV-2 vaccine candidates. Chem Commun (Camb). 2021;57(12):1474-1477. PubMed, PubMedCentralCrossRef
  25. Bellone ML, Puglisi A, Dal Piaz F, Hochkoeppler A. Production in Escherichia coli of recombinant COVID-19 spike protein fragments fused to CRM197. Biochem Biophys Res Commun. 2021;558:79-85. PubMed, PubMedCentralCrossRef

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