(152g) In Vitro Integration of Multiple Metabolic Systems for the Production of Membrane Proteins

Membrane proteins fulfill many critical
functions in living cells, making them important targets for research
and pharmaceutical development. However, the process by which these
complex proteins are expressed and folded is not well understood. In
E.coli, production of inner membrane proteins is a multi-step
process involving a wide array of metabolic processes including
transcription, translation, targeting of the ribosome to the
membrane, insertion of the translated polypeptide, intramembrane
folding and assembly, and the energy generating processes of central
metabolism and oxidative phosphorylation. We have developed a
synthetic, in vitro system that mimics all of these steps and
efficiently produces inner membrane proteins in a folded and active
form. Our cell-free production system contains a cell lysate, which
provides the enzymatic machinery needed for protein synthesis, as
well as vesicles derived from the inner membrane of E.coli,
which provide membrane-bound chaperones and the lipid bilayer
environment needed for folding and assembly of membrane proteins.
Using this system, we have produced two membrane proteins, mannitol
permease and the tetracycline pump, in high yields utilizing multiple
turnovers of the required synthesis machinery. By taking advantage
of the accessibility of our system, we have quantitatively determined
how changes in signal recognition particle (SRP), SRP receptor, and
vesicle concentrations affect overall production of active membrane
proteins.