A cell-free protein translocation system prepared entirely from a Gram-positive organism

Abstract

A cell-free protein translocation system derived exclusively from a Gram-positive bacterium is described here for the first time. Highly efficient in vitro synthesis of plasmid encoded preprolipase of Staphylococcus hyicus is accomplished by coupled transcription/translation using either a cytosolic extract of S. carnosus alone or in combination with T7-RNA-polymerase. Addition of inside-out cytoplasmic membrane vesicles of S. carnosus leads to the partial conversion (processing) of preprolipase to prolipase. In addition, as shown in a protease protection assay, a significant part of preprolipase plus prolipase is translocated in vitro into the lumen of the vesicles. Translocation of preprolipase into the membrane vesicles requires the proton-motive force and the S. carnosus SecA protein.