Recombinant protein production in Escherichia coli usually leads to accumulation of the product inside the cells. To capture the product, cells are harvested, resuspended, and lysed. However, in cases where the product is transported to the periplasm, selective disruption of the outer membrane leads to much purer crude extracts compared to complete cell lysis, as only 4-8% of the native E. coli host cell proteins are located in the periplasmic space. A variety of different strategies to enable selective release of the product from the periplasm is available. However, in most of these studies cells are harvested before they are resuspended in permeabilization agent and no differentiation between leakiness and lysis is made. Here, we tested and compared different strategies to trigger leakiness. In contrast to other studies, we performed these experiments during cultivation and quantified both leakiness and lysis. In summary, we recommend incubation with 350 mM TRIS at constant pH for several hours followed by a mild heat treatment up to 38°C to trigger leakiness with only minimal lysis. This study represents a comparative summary of different strategies to trigger E. coli leakiness and describes a solid basis for further experiments in this field.
Keywords: Escherichia coli; Lysis monitoring; Outer membrane integrity; Recombinant protein production; Selective periplasmic release.
© 2016 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.