Construction And Characterization Of TEV Protease Mutants

Tobacco etch virus protease (TEVp) is maily applied for cleavage the fusion tags owing to stringent sequence specificity. However. the production of wild type TEVp in E. coli is hampered due to poor solubility. In order to investigate the effect of amino acids and some codons on protein solublity or stability, we constructed, analyzed and comparied the TEVp variant with mutated two amino acid residues and fifteen synonymous codon variants. The results are as follows:1. The double mutant (L56V/S135G) was constructed. The expressed protein contained a N-terminal His6-tag, and a C-terminal His6-tag, or GFP tag or S-tag for detecting solubility in Escherichia coli or rapid purification.2. The soluble production of the soluble TEVp variant was monitored using GFP reporter by flow cytometer analysis. The double mutant was more soluble than the wild type TEVp, but less than the triple mutant (T17S/N68D/I77V) and the quintuple mutant (T17S/L56V/N68D/I77V/S135G). Upon increased inducible temperature, the activity level of the double mutant retained the constant, whereas that of other versions of TEVp was decreased.3. The wild type and mutated proteins were purified and characterized. The kcat/Km value of the double mutant was more than that of the wild type TEVp, but lower than that of the triple mutant and the quintuple mutant. The double mutant displayed the less thermostability than the quintuple mutant but more hermostability than the wild type protease and the triple mutant. It also exhibited most resistance to the denaturants at the concentration of 2M.4. By increased IPTG concentration up to 1 mM, most of the TEVp constructs deposited as inclusion bodies at 37℃. By coexpressed the molecular chaperones GroES and GroEL, the recovered double mutant was expressed at highest level in soluble form and had most catalytic efficiency.5. Based on the the quintuple mutant, fifteen codon variants were constructed with one or more of six rare arginine codons in the coding sequence replaced with E. coli preferred ones. All the variants contained a N-terminal His6-tag, a C-terminal His6-tag or GFP tag. The selected variants contained a N-terminal His6-tag, a C-terminal S-tag.6. The yields of the codon variants expressed in soluble and insoluble fractions were determined by analyzes of SDS-PAGE, Western blot and fluorescence intensities. The variants dispayed different expression patterns.The silent mutations affected soluble expression level and protein folding.7. The increased rare tRNA abundance in E. coli strain RosettaTM(DE3) enhanced the soluble expression level of a few of codon variants, but lowered that of other variants. Some variants expressed as inclusion bodies were resulted from the accelerated protein synthesis rate, as detected by the fused S-tag.8. The purified of selected synonymous variants displayed less activity when increased the rare tRNAs, and decreased substrate tolerance at P1 position. The variant with improved solubility and activity was selected from the codon variants libraries.In conclusion, we confirmed that generating a superior TEVp variant with the desirable solubility, stability and activity is still challenging. The synonymous rare arginine codons and tRNA abundance differently affect soluble protein, enzyme activity and specificity. By enlarging size of the libraries, the mutant with diserable solubility, activity and stability will be screened.