Extended Data Fig. 4: Purification and in vitro characterization of superPLD and PLD^WT.

a, SDS-PAGE showing His-NusA-superPLD (shown here is clone 2-48) (113 kDa) in cell lysate, His-NusA (58 kDa) being retained on the TALON beads, and superPLD (55 kDa) eluting from the beads and exhibiting high purify after size-exclusion chromatography (SEC). 6xHis-NusA-superPLD was expressed in E.coli Rosetta 2 and purified using TALON resin. HRV 3C protease was used to cleave between NusA and PLD. Black arrows indicate the bands derived from PLD. b, Ponceau stain with PLD^WT and superPLD^high purified from Rosetta 2 vs. Gami-2 (Rosetta-gami 2) strains. When expressed in the Rosetta strain, PLD^WT showed substantial degradation and lower yield. c, SDS-PAGE showing general correlation between the activity of superPLD mutants in mammalian cells and the robustness of purification from E. coli. The numbers in parentheses indicate relative PLD activity in mammalian cells as determined by IMPACT (Fig. 2d). Black arrow indicates the bands corresponding to PLD. d–f, Kinetic analysis of PLD activity. PLD^WT and a subset of superPLD mutants were diluted accordingly to adjust to equal amounts based on SDS-PAGE (d) and incubated with indicated concentration of DOPC. The purification of each PLD mutant was performed in at least three independent experiments (except for 1-4 and 1-12, which were purified in two independent experiments) with similar results, and the representative data are shown in panels a–d. Activity assays were performed using the Amplex Red Phospholipase D Assay Kit. The rates of reaction are plotted against the substrate concentration (e), and the data were fit to the Michaelis–Menten equation to obtain the kinetic parameters (f). Horizontal lines indicate average, and vertical error bars indicate standard deviation (n = 4 independent replicates). g, Thermal stability of PLD^WT and a subset of superPLD mutants spanning a wide range of activities (see Fig. 2d), indicating no clear correlation between thermal stability and PLD activity. Melting temperature (Tm) was determined in a real-time thermocycler using SYPRO Orange dye. Horizontal lines represent the mean and error bars represent the standard deviation (n = 2, 4, 6, 2, 7, 8, and 4, respectively, and n refers to the number of independent replicates). h, Chemical stability of PLD^WT and superPLD^high (2-48) purified from the Gami-2 strain, indicating increased chemical stability for superPLD. Each enzyme was incubated with indicated concentration of urea in PBS for 12 h at 37 °C, followed by an activity assay using the Amplex Red Phospholipase D Assay Kit. Relative rates of reaction compared to control samples (enzymes incubated in PBS only) are plotted. Horizontal lines indicate average, and vertical error bars indicate standard deviation (n = 4 independent replicates).

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