How to trigger periplasmic release in recombinant Escherichia coli: A comparative analysis

Recombinant protein production inEscherichia coliusually leads to accumulation ofthe 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 extractscompared to complete cell lysis, as only 4–8% of the nativeE. colihost cell proteinsare located in the periplasmic space. A variety of different strategies to enable selectiverelease of the product from the periplasm is available. However, in most of thesestudies cells are harvested before they are resuspended in permeabilization agent andno differentiation between leakiness and lysis is made. Here, we tested and compareddifferent strategies to trigger leakiness. In contrast to other studies, we performedthese experiments during cultivation and quantified both leakiness and lysis. Insummary, we recommend incubation with 350 mM TRIS at constant pH for severalhours followed by a mild heat treatment up to 38°C to trigger leakiness with onlyminimal lysis. This study represents a comparative summary of different strategiesto triggerE. colileakiness and describes a solid basis for further experiments in thisfield.