Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins

Biotechnol Prog. Jul-Aug 2005;21(4):1243-51. doi: 10.1021/bp049553u.

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