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.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
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 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
-
SEC Translocation Channels
-
SecA Proteins
-
Signal Recognition Particle / genetics
-
Signal Recognition Particle / metabolism
-
Solubility
-
Transport Vesicles / metabolism
Substances
-
Bacterial Outer Membrane Proteins
-
Bacterial Proteins
-
Escherichia coli Proteins
-
Ffh protein, E coli
-
FtsQ protein, E coli
-
FtsY protein, Bacteria
-
Membrane Proteins
-
Membrane Transport Proteins
-
Molecular Chaperones
-
Receptors, Cytoplasmic and Nuclear
-
SEC Translocation Channels
-
SecB protein, Bacteria
-
Signal Recognition Particle
-
OMPA outer membrane proteins
-
Adenosine Triphosphatases
-
SecA Proteins