A cell-free protein translocation system prepared entirely from a gram-positive organism.
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.
Schimz
K L
KL
Institut für Biotechnologie, Forschungszentrum Jülich GmbH, Germany.
Decker
G
G
Frings
E
E
Meens
J
J
Klein
M
M
Müller
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D019286:Endopeptidase K; D004792:Enzyme Precursors / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008049:Lipase / Q000096:biosynthesis / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D012697:Serine Endopeptidases / Q000378:metabolism; D013210:Staphylococcus / Q000201:enzymology / Q000378:metabolism*
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D008049:Lipase; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins