Investigation of physicochemical condition to stabilize phosphatidylcholine-liposome 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 15-30ºC, but lost the stability acutely at 35ºC. Ionic strength, given as the concentration of NaCl, also affects its stability, and a higher concentration of NaCl, i.e., more than 150 mM, 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.

The C-Terminal Domain of the Bacillus thuringiensis Cry4Ba Mosquito-Specific Toxin Serves as a Potential Membrane Anchor

“…The membrane-perturbing activity of the purified Cry4Ba-DIII truncate was assessed in comparison with its full-length toxin by measuring the toxin-induced leakage of calcein fluorescence (self-quenching fluorescent dye) from the dye-loaded lipid vesicles as previously described [ 41 ]. Large unilamellar vesicles (LUVs, mean diameter of 50–100 nm) encapsulated with 50 mM calcein were prepared from a lipid mixture (Avanti Polar Lipids, Inc., Alabaster, AL, USA) of PE, PC and Ch (10:10:1, w / w / w ) by the extrusion method according to standard procedures [ 42 ].…”