Repurposing a Bacterial Quality Control Mechanism to Enhance Enzyme Production in Living Cells
| Autor: | May N. Taw, Ka-Hei Siu, Robert J. Conrado, Larry P. Walker, Jessica C. Stark, Donna M. Gibson, Brian C. King, Matthew P. DeLisa, Jason T. Boock |
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| Rok vydání: | 2015 |
| Předmět: |
Quality Control
Protein Folding Arginine Protein Engineering medicine.disease_cause Article Cellulase Fusarium Structural Biology Escherichia coli medicine Protein biosynthesis Molecular Biology biology Protein Stability Membrane transport protein Escherichia coli Proteins Membrane Transport Proteins Protein engineering Directed evolution Solubility Biochemistry Fusariosis Mutation biology.protein Protein folding Heterologous expression Function (biology) |
| Zdroj: | Journal of Molecular Biology. 427:1451-1463 |
| ISSN: | 0022-2836 |
| Popis: | Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high biological activity. The first tier involves a genetic selection for intracellular protein stability that is based on the folding quality control mechanism inherent to the twin-arginine translocation pathway, while the second is a semi-high-throughput screen for protein function. To demonstrate the utility of this strategy, we isolated variants of the endoglucanase Cel5A, from the plant-pathogenic fungus Fusarium graminearum, whose production was increased by as much as 30-fold over the parental enzyme. This gain in production was attributed to just two amino acid substitutions, and it was isolated after two iterations through the two-tiered approach. There was no significant tradeoff in activity on soluble or insoluble cellulose substrates. Importantly, by combining the folding filter afforded by the twin-arginine translocation quality control mechanism with a function-based screen, we show enrichment for variants with increased protein abundance in a manner that does not compromise catalytic activity, providing a highly soluble parent for engineering of improved or new function. |
| Databáze: | OpenAIRE |
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