Obtaining of the human recombinant tumor necrosis factor-alpha protein in Escherichia coli cells and evaluation of its biological activity

The paper presents the key stages of obtaining recombinant human tumor necrosis factor-alpha (TNF-α) in Escherichia coli cells. The stages of constructing and cloning the gene in the pET24b(+) vector under the T7 bacteriophage promoter are described. A potential strain producing the recombinant protein based on E. coli BL21-Gold(DE3) was constructed, capable of producing a soluble form of TNF-α in an amount exceeding 30 % of the total cellular protein. Purification of the target protein was carried out using tandem ion-exchange and size-exclusion chromatography. Biological activity of the obtained protein was determined using the standard protocol for the production of biomedical cell products based on dendritic cells (DC). The viability of DC was established, which did not differ in the control and experimental groups and exceeded 90 %. At the same time, the TNF-α sample is characterized by increased expression of mature DC marker molecules CD83 and HLA-DR. The obtained data indicate high immunobiological activity in comparison with the available commercial preparation and the potential for import substitution.

1. Horiuchi T., Mitoma H., Harashima S., Tsukamoto H., Shimoda T. Transmembrane TNF-a: structure, function and interaction with anti-TNF agents. Rheumatology, 2010, vol. 49, no. 7, pp. 1215–1228. https://doi.org/10.1093/rheumatology/keq031

2. Eck M. J., Sprang S. R. The Structure of Tumor Necrosis Factor-α at 2.6 Å Resolution. Journal of Biological Chemistry, 1989, vol. 264, no. 29, pp. 17595–17605. https://doi.org/10.1016/s0021-9258(18)71533-0

3. Chan F. K. M., Chun H. J., Zheng L., Siegel R. M., Bui K. L., Lenardo M. J. A Domain in TNF Receptors That Mediates Ligand-Independent Receptor Assembly and Signaling. Science, 2000, vol. 288, no. 5475, pp. 2351–2354. https://doi.org/10.1126/science.288.5475.2351

4. Chu W. M. Tumor necrosis factor. Cancer Letters, 2013, vol. 328, no. 2, pp. 222–225. https://doi.org/10.1016/j.canlet.2012.10.01

5. Pancisi E., Granato A. M., Scarpi E., Ridolfi L., Carloni S., Moretti C., Guidoboni M. [et al.]. Stability Program in Dendritic Cell Vaccines: A “Real-World” Experience in the Immuno-Gene Therapy Factory of Romagna Cancer Center. Vaccines, 2022, vol. 10, no. 7, art. 999. https://doi.org/10.3390/vaccines10070999

6. Hanna Е., Rémuzat C., Auquier P., Toumi M. Advanced therapy medicinal products: current and future perspectives. Journal of Market Access & Health Policy, 2016, vol. 4, no. 1, art. 31036. https://doi.org/10.3402/jmahp.v4.31036

7. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, 1977, vol. 74, no. 12, pp. 5463–5467. https://doi.org/10.1073/pnas.74.12.5463

8. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. (eds.). Current protocols in molecular biology. New York, John Wiley & Sons, 2003. 4648 p.

9. Laemmli U. K. Cleavage of structural proteins during the assembly the head of bacteriophage T4. Nature, 1970, vol. 227, no. 5259, pp. 680–685. https://doi.org/10.1038/227680a0

10. Gasteiger E., Hoogland Ch., Gattiker A., Duvaud S’., Wilkins M. R., Appel R. D., Bairoch A. Protein Identification and Analysis Tools on the ExPASy Server. The Proteomics Protocols Handbook. Totowa, NJ, 2005, pp. 571–607. https://doi.org/10.1385/1-59259-890-0:571

11. Burland T. G. DNASTAR’s Lasergene Sequence Analysis Software. Bioinformatics Methods and Protocols. Vol. 132. Totowa, NJ, 1999, pp. 71–91. https://doi.org/10.1385/1-59259-192-2:71

12. Peden J. F. Analysis of Codon Usage. Ph. D. Thesis. Nottingham, 2000. 215 p.

13. Shneider C. A., Rasband W. S, Eliceiri K. W. NIH Image to ImageJ: 25 years of Image Analysis. Nature Methods, 2012, vol. 9, no. 7, pp. 671–675.

14. Gassman M., Grenacher B., Rohde B., Vogel J. Quantifying Western blots: Pitfalls of densitometry. Electrophoresis, 2009, vol. 30, no. 11, pp. 1845–1855. https://doi.org/10.1002/elps.200800720

15. Goncharov A. E., Romanova I. V., Zalutskii I. V., Prokhorov A. V., Papok V. I. Characterization of dendritic cells for immunotherapy of pancreatic cancer. Vestsі Natsyyanal’nai akademіі navuk Belarusі. Seryya medytsynskіkh navuk = Proceedings of the National Academy of Sciences of Belarus, Medical series, 2014, no. 2, pp. 4–12 (in Russian).

16. Titov L. P., Goncharov A. E., Putyrskii L. A., Koshelev S. V., Kosheleva M. I., Putyrskii Yu. L. Immunophenotype and functional properties of monocytic dendritic cells of breast cancer patients. Zdravookhranenie [Healthcare], 2010, no. 10, pp. 52–55 (in Russian).

17. Rebrova O. Yu. Statistical Analysis of Medical Data. Using the Statistica Software Package. Мoskow, Media Sfera Publ., 2003. 312 p (in Russian).

18. Riaz B., Islam S., Ryu H., Sohn S. CD83 Regulates the Immune Responses in Inflammatory Disorders. International Journal of Molecular Sciences, 2023, vol. 24, no. 3, art. 102–124. https://doi.org/10.3390/ijms24032831