Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels.

@article{Miroux1996OverproductionOP,
  title={Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels.},
  author={Bruno Miroux and John E. Walker},
  journal={Journal of molecular biology},
  year={1996},
  volume={260 3},
  pages={
          289-98
        }
}
We have investigated the over-production of seven membrane proteins in an Escherichia coli-bacteriophage T7 RNA polymerase expression system. In all seven cases, when expression of the target membrane protein was induced, most of the BL21(DE3) host cells died. Similar effects were also observed with expression vectors for ten globular proteins. Therefore, protein over-production in this expression system is either limited or prevented by bacterial cell death. From the few survivors of BL21(DE3… 

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References

SHOWING 1-10 OF 30 REFERENCES
Abundant bacterial expression and reconstitution of an intrinsic membrane-transport protein from bovine mitochondria.
TLDR
The oxoglutarate carrier inclusion bodies have been disaggregated with the detergent N-dodecanoyl-sarcosine, and the protein has been incorporated into liposomes, and these experiments remove significant obstacles to crystallization trials and to site-directed mutagenesis of the oxoglUTarate carrier.
FtsL, an Essential Cytoplasmic Membrane Protein Involved in Cell Division in Escherichia coli
TLDR
Under certain growth conditions, depletion of FtsL or expression of the largest ftsL-phoA fusion produced a variety of cell morphologies, including Y-shaped bacteria, indicating a possible general weakening of the cell wall.
Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction
TLDR
Unexpectedly, the maximum expression of either test gene has no specific effect on the relative rates of synthesis of the tRNA species that the authors studied, and there is a cumulative breakdown of rRNAs, which results in a loss of ribosomes and protein synthetic capacity.
Recent developments in heterologous protein production in Escherichia coli.
A dominant negative allele of the Escherichia coli uvrD gene encoding DNA helicase II. A biochemical and genetic characterization.
TLDR
In vivo complementation assays indicate that the mutant protein cannot substitute for the wild-type protein in methyl-directed mismatch repair, suggesting that the ATPase and/or helicase activity of helicase II is required in this repair pathway.
Transcribing of Escherichia coli genes with mutant T7 RNA polymerases: stability of lacZ mRNA inversely correlates with polymerase speed.
TLDR
It is proposed that the instability of the T7 RNA polymerase transcripts stems from the unmasking of an RNase E-sensitive site(s) between the polymerase and the leading ribosome: the faster the polymer enzyme, the longer the lag between the synthesis of this site(S) and its shielding by ribosomes, and the lower the transcript stability.
The delta- and epsilon-subunits of bovine F1-ATPase interact to form a heterodimeric subcomplex.
TLDR
It is likely that the delta- and epsilon subunits are associated in a similar manner in the bovine F1-ATPase complex, and that, like a bacterial homologue of the Delta-subunit, they interact with the gamma- and beta-subunits.
Primary structure of the alanine carrier protein of thermophilic bacterium PS3.
The stability of Escherichia coli lacZ mRNA depends upon the simultaneity of its synthesis and translation.
TLDR
It is proposed that this higher speed T7 RNA polymerase transcribes the lacZ gene approximately 8‐fold faster than the E. coli enzyme unmasks an RNase E cleavage site which is normally shielded by ribosomes soon after its synthesis when the slower E. bacteria enzyme is used.
...
...