A CELL-FREE PROTEIN TRANSLOCATION SYSTEM PREPARED ENTIRELY FROM A GRAM-POSITIVE ORGANISM

A cell-free protein translocation system derived exclusively from a Gr am-positive bacterium is described here for the first time. Highly eff icient in vitro synthesis of plasmid encoded preprolipase of Staphyloc occus hyicus is accomplished by coupled transcription/translation usin g either a cytosolic extract of S. carnosus alone or in combination wi th T7-RNA-polymerase. Addition of inside-out cytoplasmic membrane vesi cles of S. carnosus leads to the partial conversion (processing) of pr eprolipase to prolipase. In addition, as shown in a protease protectio n assay, a significant part of preprolipase plus prolipase is transloc ated in vitro into the lumen of the vesicles. Translocation of preprol ipase into the membrane vesicles requires the proton-motive force and the S. carnosus SecA protein.