Figure 4. Expression of Dictyostelium PLB in E. coli.

BL21 were transformed with control plasmid (pGEX-5X-3) and plasmid containing PLB (pGEX-5X-3-PLB). Three cultures were grown: control plasmid, PLB uninduced and PLB induced. The GST-fusion proteins were purified as described in the Experimental section, resulting in 800 μl of purified protein for each 1 litre culture. The proteins were assayed for PLB activity using the didecanoyl substrate. Briefly, 40 μl of each purified protein was incubated with 20 μl of vesicles, in a total volume of 120 μl for 60 min at 37 °C. myrARF1 and GTPγS were omitted from the assay. (A) shows GPC and (B) choline.