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A cell‐free protein translocation system prepared entirely from a Gram‐positive organism
Author(s) -
Schimz Karl-Ludwig,
Decker Gaby,
Frings Elke,
Meens Jochen,
Klein Michael,
Müller Matthias
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00180-h
Subject(s) - organism , chromosomal translocation , gram , chemistry , microbiology and biotechnology , cell , biochemistry , biology , bacteria , genetics , gene
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.