Optimization of RapR-Shp2 through flexible linker modifications. (A) Representation of the linker modifications for RapR-Shp2 showing Shp2 β-strands (gray, insertion site residues are indicated), flexible linkers (pink) and iFKBP (blue). Created using biorender.com. (B and C) Cell-free in vitro assay evaluating phosphatase activity of RapR-Shp2 Val⁴⁰⁶ insertion site with varied flexible linkers between iFKBP and the catalytic domain. Four variants depicted in Fig. 2 A were tested. Indicated constructs were immunoprecipitated and their ability to dephosphorylate purified phosphorylated paxillin was assessed in vitro. Representative blots of N = 4 independent experiments shown with quantification of Paxillin (pY¹¹⁸) blot intensity (C). Analysis of statistical significance at the 0.05 level was determined by two-tailed Student’s t test for independent pairwise comparisons of each linker with and without rapamycin. *P < 0.05. (D and E) Comparison of RapR-Shp2 and CA-Shp2 phosphatase activity. Activated RapR-Shp2 and CA-Shp2 were immunoprecipitated and incubated with purified phospho-paxillin for the indicated time. Activity assessed via immunoblot of phospho-Paxillin (pY³¹). Representative blot of N = 3 independent experiments. (E) Phosphatase activity plot showing relative change in substrate over time and exponential decay fit curve. Insert shows plot of the integrated rate equation derived to obtain relative K_m and V_max values (the slope and the intercept of the linear plot) using approach described in Orsi and Tipton (1979). RapR-Shp2 shows 1.2 increase in K_m and 2.2 increase in V_max when compared to CA-Shp2. All error bars show 90% CI.