UNIDIRECTIONAL RECONSTITUTION INTO DETERGENT-DESTABILIZED LIPOSOMES OF THE PURIFIED LACTOSE TRANSPORT-SYSTEM OF STREPTOCOCCUS-THERMOPHILUS

The lactose transport protein (LacS) of Streptococcus thermophilus was amplified to levels as high as 8 and 30% of total membrane protein in Escherichia coli and S, thermophilus, respectively, In both organisms the protein was functional and the expression levels were highest wit h the streptococcal lacS promoter, Also a LacS deletion mutant, lackin g the carboxyl-terminal regulatory domain, could be amplified to level s >20% of membrane protein, Membranes from S, thermophilus proved to b e superior in terms of efficient solubilization and ease and extent of purification of LacS; >95% of LacS was solubilized with relatively lo w concentrations of Triton X-100, n-octyl-beta-D-glucoside, n-dodecyl- beta-D-maltoside, or C(12)E(8). The LacS protein carrying a poly-histi dine tag was purified in large quantities (similar to 5 mg/liter of cu lture) and with a purity >98% in a two-step process involving nickel c helate affinity and anion exchange chromatography, The membrane recons titution of LacS was studied systematically by stepwise solubilization of preformed liposomes, prepared from E, coil phospholipid and phosph atidylcholine, and protein incorporation at the different stages of li posome solubilization. The detergents were removed by adsorption onto polystyrene beads and H+-lactose symport and lactose counterflow were measured, Highest transport activities were obtained when Triton X-100 was used throughout the solubilization/purification procedure, wherea s activity was lost irreversibly with n-octyl-beta-D-glucoside. For re constitutions mediated by n-dodecyl-beta-D-maltoside, C(12)E(8), and t o a lesser extent Triton X-100, the highest transport activities were obtained when the liposomes were titrated with low amounts of detergen t (onset of liposome solubilization), Importantly, under these conditi ons proteoliposomes were obtained in which LacS was reconstituted in a n inside-out orientation, as suggested by the outside labeling of a si ngle cysteine mutant with a membrane impermeable biotin maleimide, The results are consistent with a mechanism of reconstitution in which th e hydrophilic regions of LacS prevent a random insertion of the protei n into the membrane, Consistent with the in vivo lactose/galactose exc hange catalyzed by the LacS protein, the maximal rate of lactose count erflow was almost 2 orders of magnitude higher than that of H+-lactose symport.