Improved strains and bioprocesses for redox sensitive recombinant protein production in E. coli using two-stage dynamic control

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2022

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Abstract

Since the inception of recombinant protein expression, this technique has revolutionized our everyday lives with the number of protein products we have at our disposal ranging from household products to pharmaceuticals. E. coli was the first host used for recombinant protein expression and it has remained a mainstay expression host in biotechnology. However, the utility of E. coli as an expression host is limited by challenges that, if addressed, would make it a more suitable host for a wider variety of proteins, and thereby catalyze the translation of more protein products. Here, we address these challenges with dynamic control of the E. coli metabolism. This approach involves decoupling cell growth and protein expression and altering metabolism exclusively in the production phase. With this dynamic tool we have been able to: (1) engineer E. coli to improve the cytoplasmic solubility of redox sensitive proteins that are prone to aggregation, (2) improve the growth robustness of these engineered strains over the current state the art, (3) simply the bioprocess to purify these redox sensitive proteins, (4) identify uncharacterized redox regulation that impacts protein expression. Additionally, we provide a historical overview of biotechnological advances associated with a class of biopharmaceuticals, enzyme-based therapies, which have underutilized potential. Cumulatively, this work analyzes development trends of biologics, recognizes gaps in therapeutic and production capabilities, and provides solutions to challenges associated with redox sensitive protein expression and purification in E. coli.

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Hennigan, Jennifer (2022). Improved strains and bioprocesses for redox sensitive recombinant protein production in E. coli using two-stage dynamic control. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/25831.

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