Heterogeneous expression and characterization of a new mutant DNA-binding protein from the Thermotoga naphthophila hyperthermophilic microorganism

This paper presents data on cloning, heterogeneous expression and characterization of recombinant homologs of a DNA-binding protein contained in the Thermotoga naphthophila (TnaDBP, TnaDBP-mut) hyperthermophilic microorganism. Initially, the nucleotide sequence of the original thermostable TnaDBP protein was subjected to codon optimisation in accordance with the structural and operational features of the nucleic acid metabolism system of the Escherichia coli mesophilic bacterium subsequently used as a laboratory strain-producer. Expression vector constructs were designed to ensure efficient production of TnaDBP and TnaDBP-mut proteins in E. coli cells. Optimal conditions for the cultivation of transformed strains for the biosynthesis of the soluble form of the target protein product were selected. A trial aerobic cultivation of the microorganisms was carried out under controlled conditions. Specific features of the growth kine - tics for transformed E. coli BL21 (DE3) [pET-TnaDBP-mut] cells were studied. It shown that, under cultivation in a liquid nutrient medium, the E. coli BL21 (DE3) [pET-TnaDBP-mut] culture producing the mutant DNA-binding protein reaches the stationary growth phase 10 hours after the inducer has been administered. A scheme consisting of a few stages is proposed for purifying the obtained proteins using thermolysis. A preliminary assessment of the solubility and thermal stability of the protein according to its primary amino acid sequence was carried out. Possibilities for the practical application of recombinant variants of the thermostable TnaDBP DNA binding protein are considered. Our results evidently demo nstrate that such proteins, due to their unique physicochemical properties, present great interest from a biotechnological point of view and can be used in various industries as sources of essential L -amino acids for cultures of eukaryotic cells as a basis for enteral nutrition of farm animals, as well as a necessary component base in the development of non-viral vector systems and carrier proteins in the field of biomedicine and fundamental science.