Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins.
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.
Kuruma
Yutetsu
Y
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, FSB401, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan.
Nishiyama
Ken-Ichi
K
Shimizu
Yoshihiro
Y
Müller
Matthias
M
Ueda
Takuya
T
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D018271:Signal Recognition Particle / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D022161:Transport Vesicles / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; C068023:FtsQ protein, E coli; C087406:FtsY protein, Bacteria; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; D018160:Receptors, Cytoplasmic and Nuclear; D000069816:SEC Translocation Channels; C057842:SecB protein, Bacteria; D018271:Signal Recognition Particle; C078167:OMPA outer membrane proteins; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins