Large Complexes: Cloning Strategy, Production, and Purification

Part of the Methods in Molecular Biology book series (MIMB, volume 1615)


Membrane proteins can assemble and form complexes in the cell envelope. In Gram-negative bacteria, a number of multiprotein complexes, including secretion systems, efflux pumps, molecular motors, and pilus assembly machines, comprise proteins from the inner and outer membranes. Besides the structures of isolated soluble domains, only a few atomic structures of these assembled molecular machines have been elucidated. To better understand the function and to solve the structure of protein complexes, it is thus necessary to design dedicated production and purification processes. Here we present cloning procedures to overproduce membrane proteins into Escherichia coli cells and describe the cloning and purification strategy for the Type VI secretion TssJLM membrane complex.

Key words

Membrane protein complexes Escherichia coli T7 overexpression Protein purification 



We would like to thank M. Petiti, L. Houot, H. Célia, and D. Duché for their careful reading of the text. E.D. and R.L. are funded by the Centre National de la Recherche Scientifique, the Aix-Marseille Université, and two grants from the Agence Nationale de la Recherche (ANR-10-JCJC-1303-03 and ANR-14-CE09-0023, respectively). ED is supported by the Institut National de la Santé Et de la Recherche Médicale through a permanent research position.


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© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Laboratoire d’Ingénierie des Systèmes Macromoléculaires (LISM, UMR7255)Institut de Microbiologie de la Méditerranée (IMM), Aix-Marseille Univ and CNRSMarseille cedex 20France

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