A Tobacco Etch Virus Protease with Increased Substrate Tolerance at the P1' position
Figure 3
In vivo analysis of the P1' Specificity of the pTEV2 protease.
A) Processing of the tester constructs CFP-TDegX-RFP (plasmid encoded) was observed after induction of pTEV2 protease production (PGAL1-pTEV2 in yeast strain YCR56). Conditions as inFigure 1B.B) Quantification of the P1' Specificity of the pTEV2 protease. Decrease of full length tester construct after two hours was normalized to initial values and relative efficiency normalized to proline was calculated (cleavage efficiency = ([X]2h/[Pro]2h×100-100) ×(−1)), assuming that the recognition sequence with proline at the P1’ Position is not cleaved at all. For each construct two immunoblotting experiments were quantified. Values for constructs with Arg and Phe at the P1’ Position cleaved by the pTEV+ protease obtained at the same time are shown as reference. Yeast strains YCR56 (pTEV2 protease production) or YCT1169 (pTEV+ protease production) harboring plasmid-based constructs were used for the measurements.C) Quantification of X-RFP depletion. The RFP fluorescence was analyzed by fluorimeter measurements after induction of pTEV2 protease synthesis (upper graph, conditions as in Figure 1C) and the depletion efficiency was calculated (error bars: SEM of at least three experiments). Same constructs as in B. The difference between the arginine construct cleaved by pTEV2 and pTEV+ protease is very significant (unpaired t test; p = 0.007).