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.

recombinant protein, thermal stability, DNA binding protein, producing strain, expression, biotechnology

1. Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P. Molecular Biology of the Cell. Garland Science, 2008, 1392 p.

2. Perales C., Cava F., Meijer W.J.J., Berenguer J. Enhancement of DNA, cDNA synthesis and fidelity at high temperatures by a dimeric singlestranded DNA-binding protein. Nucleic Acids Research. 2003, vol. 31, pp. 6473–6480. DOI: 10.10 93/nar/gkg865

3. Olszewski M., Grot A., Wojciechowski M., Nowak M., Mickiewicz M., Kur J. Characterization of exceptionally thermostable single-stranded DNAbinding proteins from Thermotoga maritima and Thermotoga neapolitana. BMC Microbiology. 2010, vol. 10, pp. 260–360. DOI: 10.1186/1471-2180-10-260

4. Cernooka E., Rumnieks J., Tars K., Kazaks A. Structural Basis for DNA Recognition of a Singlestranded DNA-binding Protein from Enterobacter Phage Enc34. Scientific Reports. 2017, vol. 7, issue 1, pp. 15529–15539. DOI: 10.1038/s41598-017-15774-y

5. Kur J., Olszewski M., Długołecka A., Filipkowski P. Single-stranded DNA-binding proteins (SSBs) – sources and applications in molecular biology. Acta Biochimica Polonica. 2005, vol. 52, issue 3, pp. 569–574.

6. Grishin D.V., Pokrovskaya M.V., Podobed O.V., Gladilina J.A., Pokrovskii V.S., Aleksandrova S.S., Sokolov N.N. Prediction of protein thermostability from their primary structure: the current state and development factors. Biomeditsinskaya khimiya. 2017, vol. 63, no. 2, pp. 124–131. (In Russian). DOI: 10.18097/PBMC20176302124

7. Sterner R., Liebl W. Thermophilic adaptation of proteins. Critical Reviews in Biochemistry and Molecular Biology. 2001, vol. 36, issue 1, pp. 39–106. DOI: 10.1080/20014091074174

8. Ming D., Hellekant G. Brazzein, a new highpotency thermostable sweet protein from Pentadiplandra brazzeana B. FEBS Letters. 1994, vol. 355, issue 1, pp. 106–108.

9. Grishin D.V., Gudov V.P., Sergienko O.V., Lunin V.G., Kharchenko P.N. Creation of a protein vector construct including an SSBTne DNA-binding domain and VirD2 nuclear localization signal. Russian Agricultural Sciences. 2008, vol. 34, issue 5, pp. 329–331. DOI: 10.3103/S1068367408050145

10. Cuadros C., Lopez-Hernandez F.J., Dominguez A.L, McClelland M., Lustgarten J. Flagellin fusion proteins as adjuvants or vaccines induce specific immune responses. Infection and Immunity. 2004, vol. 72, isssue 5, pp. 2810–2816. DOI: 10.1128/IAI.72.5.2810-2816.2004

11. Rizzuto R., Brini M., Pizzo P., Murgia M., Pozzan T. Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Current Biology. 1995, vol. 5, issue 6, pp. 635–642. https://doi.org/10.1016/S0960-9822(95)00128-X

12. Grishin D.V., Gladilina Y.A., Aleksandrova S.S., Pokrovskaya M.V., Podobed O.V., Pokrovskii V.S., Zhdanov D.D., Sokolov N.N. Creation of thermostable polypeptide cassettes for amino acid balancing in farm animal rations. Applied Biochemistry and Microbiology. 2017, vol. 53, no. 6, pp. 688–698. DOI: 10.1134/S0003683817060072

13. Grishin D.V., Podobed O.V., Gladilina Yu.A., Pokrovskaya M.V., Aleksandrova S.S., Pokrovskii V.S., Sokolov N.N. Bioactive proteins and peptides: current state and new trends of practical application in the food industry and feed production. Voprosy pitaniya. 2017, vol. 86, no. 3, pp. 19–31. (In Russian)

14. Altschul S.F., Madden T.L., Schaffer A.A., Zhang J., Zhang Z., Miller W., Lipman D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research. 1997, vol. 25, no. 17, pp. 3389–3402. DOI: 10.1093/nar/25.17.3389

15. Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., Higgins D.G. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research. 1997, vol. 25, no. 24, pp. 4876–4882.

16. Kibbe W.A. OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Research. 2007, vol. 35, pp. 43–46. DOI: 10.1093/nar/gkm234

17. Ku T.H., Lu P.Y., Chan C.H., Wang T.S., Lai S.Z., Lyu P.C., Hsiao N.W. Predicting melting temperature directly from protein sequences. Computation Biology and Chemistry. 2009, vol. 33, issue 6, pp. 445–450. DOI: 10.1016/j.compbiolchem.2009.10.002

18. Diaz A., Tomba E., Lennarson R., Richard R., Bagajewicz M., Harrison R.G. Prediction of protein solubility in Escherichia coli using logistic regression. Biotechnology and Bioengineering. 2010, vol. 105, issue 2, pp. 374–383. htts://doi.org/10.1002/bit.22537

19. Drury L. Transformation of bacteria by electroporation. Methods in Molecular Biology. 1996, vol. 58., pp. 249–256. 20. Gibson D.G. Enzymatic assembly of overlapping DNA fragments. Methods in Enzymology. 2011, vol. 498, pp. 349–361. DOI: 10.1016/B978-0-12-385120-8.00015-2

20. Yadav P., Yadav A., Garg V., Datta T.K., Goswami S.L., De S. A novel method of plasmid isolation using laundry detergent. Indian Journal of Experimental Biology. 2011, vol. 49, issue 7, pp. 558–560.

21. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 1976, vol. 72, issue 1–2, pp. 248–254. http://dx.doi.org/10.1016/0003-2697(76)90527-3

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

23. Yang C., Xu Y., Jia R., Li P., Zhang L., Wang M., Zhu D., Chen S., Liu M., Yin Z., Cheng A. Prokaryotic expression of a codon-optimized capsid gene from duck circovirus and its application to an indirect ELISA. Journal of Virological methods. 2017, vol. 247, pp. 1–5. DOI: 10.1016/j.jviromet.2017.05.003

24. Tanaka M., Tokuoka M., Gomi K. Effects of codon optimization on the mRNA levels of heterologous genes in filamentous fungi. Applied Microbiology and Biotechnology. 2014, vol. 98, no. 9, pp. 3859–3867. DOI: 10.1007/s00253-014-5609-7

25. Gustafsson C., Govindarajan S., Minshull J. Codon bias and heterologous protein expression. Trends in Biotechnolody. 2004, vol. 22, issue 7, pp. 346–353. DOI: 10.1016/j.tibtech.2004.04.006

26. Murashima K., Kosugi A., Doi R.H. Solubilization of cellulosomal cellulases by fusion with cellulose‐binding domain of noncellulosomal cellulase engd from Clostridium cellulovorans. Proteins. 2003, vol. 50, issue 4, pp. 620–628. DOI: 10.1002/prot.10298

27. Yuan H., Yang X., Hua Z.C. Optimization of expression of an annexin V-hirudin chimeric protein in Escherichia coli. Microbiological Research. 2004, vol. 159, issue 2, pp. 147–156. DOI: 10.1016/j.micres.2004.02.002

28. Costa S., Almeida A., Castro A., Domingues L. Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system. Frontiers in Microbiology. 2014, vol. 5, 20 p. DOI: 10.3389/fmicb.2014.00063