Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain

Journal of Bioscience and Bioengineering
Robin LammJochen Büchs

Abstract

During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is stron...Continue Reading

Citations

Jun 4, 2020·Microbial Cell Factories·Marcos Gil-GarciaSalvador Ventura
Jul 12, 2020·Applied Microbiology and Biotechnology·Vera D JägerUlrich Krauss

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