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  • Title: TolAIII co-overexpression facilitates the recovery of periplasmic recombinant proteins into the growth medium of Escherichia coli.
    Author: Wan EW, Baneyx F.
    Journal: Protein Expr Purif; 1998 Oct; 14(1):13-22. PubMed ID: 9758746.
    Abstract:
    Overproduction of the third topological domain of the transmembrane protein TolA (TolAIII) in the periplasm of Escherichia coli confers a "leaky" phenotype to host cells by disrupting the integrity of the outer membrane and causing periplasmic proteins to leach into the growth medium. To examine the physiological consequences of TolAIII overexpression in more detail and assess the usefulness of this strategy for the release of periplasmic recombinant proteins into the extracellular fluid, we constructed a ColE1-compatible plasmid encoding a fusion between the ribose binding protein signal sequence and TolAIII under T7lac transcriptional control. About half of the total TolAIII synthesized in IPTG-induced cells aggregated in a precursor form in the cytoplasm. However, the majority of the mature protein was soluble and located in the extracellular fluid. TolAIII-overproducing cultures exhibited only slight growth defects upon entry into stationary phase but underwent extensive lysis when treated with 0.1% (w/v) SDS, and were unable to divide when supplemented with 0.02% SDS. The loss of outer membrane integrity resulted in long-term damage since cell viability was reduced by three orders of magnitude compared to control or uninduced cells. Overexpression of TolAIII did not significantly interfere with the translocation and processing of a plasmid-encoded fusion between the OmpA signal sequence and TEM-beta-lactamase but led to the release of most periplasmic proteins and 90% of the active enzyme into the extracellular fluid. Although the total levels of beta-lactamase accumulation in TolAIII-overproducing cultures was only 1.5- to 2-fold less than in control cells, the formation of periplasmic inclusions bodies was completely suppressed. A threshold concentration of TolAIII was necessary for efficient release of periplasmic proteins since the viability and detergent sensitivity of uninduced cells was comparable to that of control cultures and 80% of the beta-lactamase synthesized remained confined to the periplasm.
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