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 in Escherichia 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.
KeywordsStructural biology Membrane protein Recombinant expression Escherichia coli T7 RNA polymerase Bibliographic analysis
This work was supported by the Agence National de La Recherche (ANR MIT-2M, 2010 BLAN1518), the Centre National de la Recherche Scientifique, and by the “Initiative d’Excellence” programme from the French State (Grant “DYNAMO”, ANR-11-LABEX-0011–01).
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