Cell-free expression profiling of E. coli inner membrane proteins.
The high versatility and open nature of cell-free expression systems offers unique options to modify expression environments. In particular for membrane proteins, the choice of co-translational versus post-translational solubilization approaches could significantly modulate expression efficiencies and even sample qualities. The production of a selection of 134 alpha-helical integral membrane proteins of the Escherichia coli inner membrane proteome focussing on larger transporters has therefore been evaluated by a set of individual cell-free expression reactions. The production profiles of the targets in different cell-free expression modes were analyzed independently by three screening strategies. Translational green fluorescent protein fusions were analyzed as monitor for the formation of proteomicelles after cell-free expression of membrane proteins in the presence of detergents. In addition, two different reaction configurations were implemented and performed either by robotic semi-throughput approaches or by individually designed strategies. The expression profiles were specified for the particular cell-free modes and overall, the production of 87% of the target list could be verified and approximately 50% could already be synthesized in preparative scales. The expression of several selected targets was up-scaled to milliliter volumes and milligram amounts of production. As an example, the flavocytochrome YedZ was purified and its sample quality was demonstrated.
Schwarz
Daniel
D
Center for Biomolecular Magnetic Resonance, Goethe-University of Frankfurt/Main, Frankfurt/Main, Germany.
Daley
Daniel
D
Beckhaus
Tobias
T
Dötsch
Volker
V
Bernhard
Frank
F
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D008969:Molecular Sequence Data; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C043426:inner membrane protein, E coli