In vivo synthesis of membrane proteins


Our goal is to improve the performances of expression of bacteria strains for overexpression of membrane proteins and their insertion in the bacteria membrane.


Previous work :

In the bacteria strain BL21(DE3), the induction of the RNA polymerase of phage T7 expression by adding IPTG leads very (too) quickly the transcription of the target gene from the expression plasmid. In a couple of minutes, the RNA levels of the target gene reach the level of messenger RNAs. The bacterial growth stops immediately, the immense majority of the bacteria looses the expression plasmid and/or die. However, to a non negligible frequency (10-4) some mutant bacteria conserve the capacity to express the target gene and continue to grow. These bacterial clones are isolated by simple dilution of the culture on petri dishes containing the inductor of the expression system. We observe colonies of normal sizes and small colonies, these latter being highly fluorescent under UVs. Applied to the bacterial strain BL21(DE3) this method has led to the isolation of the strains C41(DE3) and C43(DE3).

In some conditions of culture and with the b subunit of E. coli's ATP synthase, the strain C43(DE3) has shown extraordinary adaptation capacities to the production of a hydrophobic membrane protein, by synthetizing more lipids, lipids that form a complex network of intracellular membranes, as shown in the electron microscopy image below (Arechaga, Miroux et al. FEBS, 2000).



Publications

  • Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. B. Miroux J. E. and Walker. J. Mol. Biol. 260 : 289-298 (1996)

  • Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase. I. Arechaga, B. Miroux, S. Karrasch, R. Huijbregts, B. de Kruijff, M. J. Runswick, J. E. and Walker. FEBS Lett. 482 : 215-219 (2000)


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