OPTIMIZATION OF METHODS FOR PREPARATION OF RECOMBINANT TOBACCO ETCH VIRUS PROTEASE FROM Escherichia coli CELLS

The catalytic domain of the nuclear inclusion protein of the tobacco etch virus protease, TEVp, is used for the cleavage of artificial fusion polypeptides. However, the production of a recombinant enzyme has certain difficulties, such as a low yield of the product and its low solubility in physiological solutions. The aim of the study was to optimize the methods of producing a recombinant enzyme TEVp from E. coli producing cells. Material and methods. The studies were carried out on E. coli cells st. BL21 (DE3). The enzyme was synthesized by cells in the form of a fusion polypeptide with maltose-binding protein (MBP), followed by self-cleavage. Biomass production was carried out under various conditions: a change in the temperature regime, the time of incubation of cells with an inducer, the concentration of the inducer and the growth phase of the culture with the addition of an inducer. The enzyme was isolated under native conditions and with an increased concentration of sodium chloride by affinity chromatography. The enzyme activity was tested on chimeric recombinant human apolipoprotein A-I (~33.4 kDa). Results and discussion. The results of the study showed that a significant influence on the final yield of the enzyme was provided by the growth phase of the culture when the inducer was added. Optimal conditions for obtaining biomass were found as follows: incubation temperature with an inducer 30 °C; incubation time 4 hours; inducer concentration 200 μM; optical density with inducer addition 2.0–2.5 optical units per ml. Sodium chloride concentration in the buffer solution during isolation of the protein was 150 mM. The yield of the enzyme under these conditions reached 50 mg from a liter of cell culture. A similar yield of the enzyme was obtained using the method of auto-induction of cell culture. In all cases, enzymes retained their activity. Conclusion. It was shown that the greatest influence on the yield of the recombinant enzyme from E. coli producing cells strain BL21 (DE3) in the expression vector pD441-MBP under the regulation of the bacteriophage promoter gene «T5» was exerted by the growth phase of the cell culture at the time of gene expression launch.

Blommel P.G., Fox B.G. A combined approach to improving large-scale production of tobacco etch virus protease // Protein Expr. Purif. 2007. 55. (1). 53-68.

Cabrita L.D., Gilis D., Robertson A.L., Dehouck Y., Rooman M., Bottomley S.P. Enhancing the stability and solubility of TEV protease using in silico design // Protein Sci. 2007. 16. (11). 2360-2367.

Fang L., Jia K.Z., Tang Y.L., Ma D.Y., Yu M., Hua Z.C. An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization // Protein Expr. Purif. 2007. 51. (1). 102-109.

Kapust R. B., Waugh D. S. Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused // Protein Sci. 1999. 8. (8). 1668-1674.

Kapust R.B., Tozser J., Fox J.D., Anderson D.E., Cherry S., Copeland T.D., Waugh D.S. Tobacco etch virus protease: Mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency // Protein Eng. 2001. 14. (12). 993-1000.

Lucast L.J., Batey R.T., Doudna J.A. Large-scale purification of a stable form of recombinant tobacco etch virus protease // Biotechniques. 2001. 30. (3). 544–546.

Nautiyal K., Kuroda Y. A SEP tag enhances the expression, solubility and yield of recombinant TEV protease without altering its activity // N. Biotechnol. 2018. 42. 77-84.

Ryabchenko A.V., Kotova M.V., Polyakov L.M. Construction protease producers of tobacco etch virus. International Journal of Applied And Fundamental Research. 2015. (12-5). 859-862. [In Russia]

Ryabchenko A.V., Kotova M.V., Tverdohleb N.V., Knyazev R.A., Polyakov L.M. Production and analysis of biological properties of recombinant human apolipoprotein A-I // Bull. Exp. Biol. Med. 2015. 160. (1). 129-133.

Studier F.W. Protein production by auto-induction in high density shaking cultures // Protein Expr. Purif. 2005. 41. (1). 207-234.

Sun C., Liang J., Shi R., Gao X., Zhang R., Hong F., Yuan Q., Wang S. Tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives // Protein Expr. Purif. 2012. 82. (1). 226-231.

Van den Berg S., Lofdahl P., Hard T., Berglund H. Improved solubility of TEV protease by directed evolution // J. Biotechnol. 2006. 121. (3). 291-298.

Zou Z., Cao L., Zhou P., Su Y., Sun Y., Li W. Hyper-acidic protein fusion partners improve solubility and assist correct folding of recombinant proteins expressed in Escherichia coli // J. Biotechnol. 2008. 135. (4). 333-339.

Polyakov L.M. – doctor of medical sciences, professor, head of the laboratory, e-mail plm@niibch.ru