Membrane Protein Production in Escherichia coli: Overview and Protocols

Structural biology of membrane proteins is hampered by the difficulty to express and purify them in a large amount. Despite recent progress in biophysical methods that have reduced the need of biological materials, membrane protein production remains a bottleneck in the field and will require further conceptual and technological developments. Among the unique 424 membrane protein structures found in protein databases, about half of them come from proteins produced inEscherichia coli. In this chapter, we have reviewed the existing bacterial expression systems. The T7 RNA polymerase-based expression system accounts for up to 62 of solved heterologous membrane protein structures. Among the dozen of bacterial hosts available, the mutant hosts C41(DE3) and C43(DE3) have contributed to half of the integral membrane protein structures that were solved after production using the T7 expression system. After a general introduction on this expression system, the protocol section of this chapter provides detailed protocols to select bacterial expression mutant hosts and to optimize culture conditions.