Optimization of the Production of Soluble Recombinant TEV Protease in Two E. coli Strains.

Background: The low solubility of Tobacco Etch Virus (TEV) protease, a functional enzyme that cleaves protein tags without significant modification in its sequence, is one of the most important limitations of this enzyme. In this study, the aim was to increase the solubility of TEV by changing the expression conditions and designing lysis buffer with various solubilizing agents to improve its solubility.

Methods: Escherichia coli (E. coli) BL21 (DE3) and E. coli origami harboring wild type TEV-pKR793 and mutant N23F TEV-pKR793 plasmids were used for the expression. Response surface methodology was used to determine the best culture conditions (IPTG concentration, incubation time and incubation temperature) of soluble expression. Furthermore, eight different solubilizing agents were added separately to the lysis buffer to check their effect on the protein solubility.

Results: The production of soluble N23F in E. coli BL21 (DE3) was two-folds more than the wild type and the inclusion body formation in the mentioned form was diminished as about 25% in comparison to the wild type. Finally, betaine had the most effects for enhancing the soluble expression of N23F in both host cells. For the wild type, sodium selenite, xylitol, and glycine showed the most effects on soluble production.

Conclusion: The solubility of the mutant form of TEV protease increased in E. coli BL21 (DE3) compared to its wild form. Also, using additives such as betaine to the lysis buffer, increased the solubility of N23F in E. coli BL21 (DE3) and origami strains.