Investigation of physicochemical condition to stabilize phosphatidylcholineliposome enclosing fluorescent calcein and its exploitation for analysis of pore formation with Cry1A toxins of Bacillus thuringiensis

Abstract

Liposome was prepared using phosphatidylcholine (PC), and calcein, a fluorescent chemical, was simultaneously enclosed within the liposome (PC-Lipo). The stability of PC-Lipo in its ability to retain calcein was evaluated under various conditions. PC-Lipo lost stability at pH 6 and pH 11-13, but was stable in the range of pH 8.3-10. PC-Lipo was stable in the temperature range of 15-30°C, but lost the stability acutely at 35°C. Ionic strength, given as the concentration of NaCl, also affects its stability, and a higher concentration of NaCl, i.e., more than 150mM, induced a higher leak of calcein from PC-Lipo. The optimal conditions to achieve stable PC-Lipo were employed to characterize the differences in pore formation with Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry1Ac toxins. These toxins were reacted with PC-Lipo under these optimal conditions, and the affinity and maximum speed of Cry1Ab to form pores on PC-Lipo was shown to be highest. Here we show these conditions and evidence of the usefulness of PC-Lipo to investigate the mode of action of Cry1A toxins.