FEBS Letters | |
A cell‐free protein translocation system prepared entirely from a Gram‐positive organism | |
Frings, Elke1  Klein, Michael1  Decker, Gaby1  Müller, Matthias2  Meens, Jochen1  Schimz, Karl-Ludwig1  | |
[1] Institut für Biotechnologie, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany;Institut für Physikalische Biochemie, Ludwig-Maximilians-Universität München, Schillerstr 44, D-80336 München, Germany | |
关键词: Protein secretion; Gram-positive bacteria; Cell-free translocation system; SecA; Lipase; Staphylococcus carnosus; lip; S. hyicus; lipase encoding gene; ppL; preprolipase; pL; prolipase; mL; mature lipase; INV; inside-out cytoplasmic membrane vesicles; DCCD; dicyclohexyl carbodiimide; FCCP; carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone; IPTG; isopropyl-1-thio-β-d-galactoside; TeaOAc; triethanolamine acetate; PVDF; polyvinylidene difluoride; SDS-PAGE; polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate; | |
DOI : 10.1016/0014-5793(95)00180-H | |
学科分类:生物化学/生物物理 | |
来源: John Wiley & Sons Ltd. | |
【 摘 要 】
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.
【 授权许可】
Unknown
【 预 览 】
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