Optimization And Functional Analysis Of The TEV Protease

Tobacco etch virus protease(TEVp)is widely utilized for cleavage the fusion tags owing to its stringent sequence specificity.Previously,we constructed the TEVp5 M codon variant with increased yield but decreased specific activity.To further improve protein folding and increase the protein expression level of the TEVp,we combined different approaches to obtain new TEV protease with high yield and activity.Cleavage of different fusions absorbed with the corresponding resin using the constructed fusion protein.The results are as follows:1.Three variants were constructed.Based on the TEVp5 M codon variant,we created three variants with mutations of K45 F,E106G and K45F/E106 G.2.Expression level and activity of TEVp variants were investigated.Qualitative analysis of TEVp variants in soluble were determined by SDS-PAGE and quantitative analysis of TEVp variants in soluble showed that as comparison to the TEVp5 M codon variant,soluble yield of E106 G variant in E.coli BL21(DE3)was increased by 28%,contrary to that of other two variants K45 F and K45F/E106 G by GFP fluorescence intensities.Qualitative and quantitative analysis of the purified fusion protein H6GST-tevS-eDAL as a TEVp substrate showed that activity of E106 G variant on cleaving the designed protein substrate was raised by 32%.Other two variants including K45 F and K45F/E106 G were less active than the TEVp5 M codon variant.3.Five TEVpE105 G fusion proteins were constructed.We chose four protein tags including GroEL,GroES,DnaK and MBP to fuse with the E106 G variant at N-terminus respectively.The TEVp recognition sequences is placed at the fusion junction of the fused tag and target protein.InfB(1–21)sequence is directly attached to the TEVp5ME106 G variant.4.Expression level and activity of TEVp mutant in fusion protein were assessed.DnaK and MBP effectively enhanced soluble production of the TEVp construct and InfB(1–21)sequence decreased solubility of the protein than the His6-tag in E.Coli BL21(DE3)but GroEL and MBP significantly increased the activity of TEVp mutant.5.Supplying rare tRNAs on function of the fusion tag was examined.In E.coli strain RosettaTM(DE3),InfB(1–21)sequence and MBP effectively enhanced soluble production of the TEVp.InfB(1–21)sequence and GroEL increased highest cleavage activity of TEVp mutant.6.Effect of the TEVp recognition sequences on fusion protein was analyzed.The tevS in the fusion protein MBP-tevS-TEVp-H6 was deleted to generate the fusion MBP-TEVp-H6.We found that deletion of the tevS did not affect soluble expression levels in two in E.coli strains correspondingly but significantly enhanced the activity in the RosettaTM(DE3).7.Mutant protein with different fusion tag were purified respectively.We analyzed purification and specific activity of different TEVp constructs and determined efficiency of on-resin cleavage of the five fusion proteins.In conclusion,we confirmed that further amino acid mutations in improving protease soluble were influenced by other mutations.Certain fusion tags on improving protein production and quality is dependent with rare tRNAs abundance.In E.coli strain RosettaTM(DE3),We first identified that GroES faintly increased protein solubility of the TEVp.The current research shows that the strain selection is important for optimization of the fusion tags.Even with assistance of the fusion tag,desirable production and quality of TEVp construct are not combined in the purified TEVp constructs.The constructed fusion protein of TEVp mutant for on-resin cleavage of partial fusion proteins made certain target proteins purified in single step.