Tag Archives: recombinant proteins

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;
*e-mail: kolibo@biochem.kiev.ua

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.

Changes in proHB-EGF expression after functional activation of the immune system cells

T. O. Chudina1,2, A. J. Labintsev1, S. I. Romaniuk1, D. V. Kolybo1, S. V. Komisarenko1

1Palladin Institute of Biochemistry, National Academy  of Sciences of Ukraine, Kyiv;
2ESC Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Ukraine;
e-mail: kolibo@biochem.kiev.ua

The level of proHB-EGF expression on J774, Raji, KG-1 cells derived from different types of human and mouse immune system cells under the standard in vitro culture conditions and during functional activation of these cells was investigated. Changes in the proHB-EGF expression on the cell surface were found to depend on the density of cell population, the content of fetal bovine serum in the culture medium, the effect of mitogenic factors – bacterial lipopolysaccharide, an inactive full-size form of diphtheria toxin (CRM197) and recombinant soluble HB-EGF – rsHB-EGF. The results obtained are important for the understanding of the functional role of proHB-EGF receptor on the surface of macrophage-like cells and B lymphocytes and indicate the involvement of this receptor in immune response regulation in an organism.