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

FEBS Letters

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