| Method |
The plasmid pTrcHisUhpT (Tamai et al 1997, E. Tamai, M.C. Fann, T. Tsuchiya and P.C. Maloney, Purification of UhpT, the sugar phosphate transporter of Escherichia coli,) was modified to contain an EcoRI cleavage site downstream from the 10-His tag. Mutagenesis was performed using the Chameleon Double Stranded, Site-Directed Mutagenesis kit (Stratagene, CA). Luria broth cultures containing 50 µg/ml ampicillin were incubated for 13 to 16 hours at 37 °C, diluted 100-fold into fresh broth, and propagated to an A600 nm of about 1.5. Induction of 10-His-AqpZ was achieved by addition of 1 mM IPTG for two hours at 37 °C before centrifugation (15 minutes at 9000 g). Solubilization of AqpZ. Purified 10-His-AqpZ was reconstituted into proteoliposomes. The osmotic behavior of reconstituted proteoliposomes and control liposomes was analyzed by following the light scattering of the preparation in a stopped-flow apparatus (SV.17MV, Applied Photophysics) with a measured dead time of 0.7 ms. The osmotic water permeability (Pf) was calculated using the following expression: Pf=k/(S/V0)×Vw×?osm where S/V0 is the vesicle surface area to initial volume ratio, Vw is the partial molar volume of water (18 cm^3), and ?osm is the difference in osmolarity between the intravesicular and extravesicular aqueous solutions (usually 300 mos mol). The radius of proteoliposomes was measured by electron microscopy. |