Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach
Fig 3
a) Reducing SDS-PAGE analysis of the generated mature proteases. b) Far-UV CD spectra of mature proteases in the range 190 to 250 nm. c) MALDI-TOF MS spectra of mature proteases as shown in the SDS-PAGE (panel a). d) Peptide mass finger printing analyses after trypsin in-gel digestion of I86L and WT (TS) mature protease. e) Theoretical mass and sequence of the N-terminus of expected cleavages upto 7 residue downstream of the N-terminus of mature papain from plant latex. Sequences marked in red and green correspond to the peaks marked by same colored arrows in panel d. Red and green arrows represent high and low intensity peaks respectively in panel d. f) Sequence of the recombinant WT (TS); yellow, blue and black colour represent vector tag, pro-peptide and mature domain sequence respectively. Red and green arrows indicate cleavages corresponding to the peptide masses indicated in the same color in MS experiment (panels d and e). The sequences which matched with mature domain obtained from MS/MS analyses are underlined.