Fig. 5
Identification of a de novo peptide that binds Cyclophilin B. a Bacterial two-hybrid assay to identify de novo interacting partner against CYPB from dicodon library. Streaks of bacterial two-hybrid experiments between DIEL-pBTnn and CYPBpTRGqq on X-gal indicator plate. All streaks are labeled to represent genes cloned in pBTnn and pTRGnn. CFP10pTRGnn/Emptyp BTnn and CFP10pTRGnn/ESAT6pBTnn signify the negative and positive controls, respectively. b Liquid β-galactosidase assay to estimate the relative enzymatic activity in Miller’s unit (M.U.) of the interaction between identified de novo dicodon library interacting partner (CDP3) and CYPB. The graph is the average of three independent assays with error bars representing the standard deviation. All values were tested for significance using a two-tailed unpaired Student’s t-test with Welch’s correction. ***P < 0.001. c, d Bacterial three-hybrid assay to monitor the disruption of PfRhopH3-C/CypB interaction by CDP3 in vivo. X-Gal indicator plate without l-arabinose and X-gal indicator plate with l-arabinose. Test streaks: Triple co-transformants containing PfRhopH3CpBTqq, CYPBpTRGqq, and CDP3pMTSA; control streaks: triple co-transformants containing PfRhopH3-CpBTqq, CYPBpTRGqq, and empty pMTSA. e l-arabinose gradient liquid β-galactosidase assay. Relative β-galactosidase activity Miller’s unit (M.U.) of the triple co-transformants containing PfRhopH3-CpBTqq, CYPBpTRGqq and empty pMTSA (red line, control) and triple co-transformants containing PfRhopH3CpBTqq, CYPBpTRGqq, and CDP3pMTSA (black line, test), is plotted against a range of l-arabinose concentrations. The graph is the average of three independent assays and the standard deviation is represented as the error bars. Multiple unpaired t-tests to compare enzyme activity of each triple co-transformant across individual l-arabinose concentrations were used. Statistical significance was determined using the unpaired t-test with Welch’s correction. **P < 0.01 was considered significant. f Western blot of triple co-transformants R1 E. coli whole-cell lysates shown in e, in order to analyze the concomitant expression of CDP3 protein with increasing concentrations of l-arabinose
