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.