How to trigger periplasmic release in recombinantEscherichia coli: A comparative analysis
| Autor: | Christoph Herwig, Oliver Spadiut, David J. Wurm, Christoph Slouka, Tadej Bosilj |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0106 biological sciences
0301 basic medicine Tris Recombinant escherichia coli Environmental Engineering Lysis Bioengineering Periplasmic space Biology medicine.disease_cause 01 natural sciences 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology chemistry Biochemistry 010608 biotechnology Recombinant protein production medicine Bacterial outer membrane Incubation Escherichia coli Biotechnology |
| Zdroj: | Engineering in Life Sciences. 17:215-222 |
| ISSN: | 1618-0240 |
| Popis: | 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. Summarizing, 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. This article is protected by copyright. All rights reserved |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|