PMID:16080708
| Citation |
Kuruma, Y, Nishiyama, K, Shimizu, Y, Müller, M and Ueda, T Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins. Biotechnol. Prog. 21:1243-51 |
|---|---|
| Abstract |
By combining translation and membrane integration/translocation systems, we have constructed a novel cell-free system for the production of presecretory and integral membrane proteins in vitro. A totally defined, cell-free system reconstituted from a minimal number of translation factors was supplemented with urea-washed inverted membrane vesicles (U-INVs) prepared from Escherichia coli, as well as with purified proteins mediating membrane targeting of presecretory and integral membrane proteins. Initially, efficient membrane translocation of a presecretory protein (pOmpA) was obtained simply by the addition of only SecA and SecB. Proteinase K digestion clearly showed the successful translocation of pOmpA inside the vesicles. Next, integration of an inner membrane protein (MtlA) into U-INVs was achieved in the presence of only SRP (Ffh) and SR (FtsY). Finally, a membrane protein possessing a large periplasmic region (FtsQ) and therefore requiring both factors (SRP/SR and SecA/SecB) for membrane integration/translocation was also shown to be integrated correctly in this cell-free system. Thus, our novel cell-free system provides not only an efficient strategy for the production of membrane-related proteins but also an improved platform for the biological study of protein translocation and integration mechanisms. |
| Links |
PubMed Online version:10.1021/bp049553u |
| Keywords |
Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/metabolism; Bacterial Outer Membrane Proteins/genetics; Bacterial Outer Membrane Proteins/metabolism; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Cell-Free System; Escherichia coli; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Membrane Proteins/genetics; Membrane Proteins/metabolism; Membrane Proteins/secretion; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Molecular Biology/methods; Molecular Chaperones/chemistry; Molecular Chaperones/metabolism; Protein Biosynthesis; Protein Transport; Receptors, Cytoplasmic and Nuclear/genetics; Receptors, Cytoplasmic and Nuclear/metabolism; Signal Recognition Particle/genetics; Signal Recognition Particle/metabolism; Solubility; Transport Vesicles/metabolism |
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