Large complexes: Cloning strategy, production and purification

Eric Durand (1) , Roland Lloubès (2)

1 PARCC - UMR-S U970 - Paris-Centre de Recherche Cardiovasculaire
2 LISM - Laboratoire d'ingénierie des systèmes macromoléculaires

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 or pilus assembly machines comprise proteins from the inner and the outer membrane. Beside the structures of isolated soluble domains, only 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 E. coli cells and describe the cloning and purification strategy for the Type VI secretion TssJLM membrane complex.

Keywords: membrane protein complexes; E coli; T7 overexpression; protein purification

Domains: Life Sciences [q-bio]; Biochemistry, Molecular Biology

Eric Durand, Roland Lloubès. Large complexes: Cloning strategy, production and purification. Methods in Molecular Biology: Bacterial protein secretion systems, 2017, ⟨10.1007/978-1-4939-7033-9_24⟩. ⟨hal-01788569⟩