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Two-Tiered Selection and Screening Strategy to Increase Functional Enzyme Production in E. coli

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Insoluble Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2406))

Abstract

Development of recombinant enzymes as industrial biocatalysts or metabolic pathway elements requires soluble expression of active protein. Here we present a two-step strategy, combining a directed evolution selection with an enzyme activity screen, to increase the soluble production of enzymes in the cytoplasm of E. coli. The directed evolution component relies on the innate quality control of the twin-arginine translocation pathway coupled with antibiotic selection to isolate point mutations that promote intracellular solubility. A secondary screen is applied to ensure the solubility enhancement has not compromised enzyme activity. This strategy has been successfully applied to increase the soluble production of a fungal endocellulase by 30-fold in E. coli without change in enzyme specific activity through two rounds of directed evolution.

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Acknowledgments

This material is based upon work supported by a USDA NIFA Grant # 2009-02202 (D.M.G and M.P.D.), an NSF GK-12 “Grass Roots” Fellowship under Grant # DGE-1045513 (J.T.B.), and Miami University Assistant Professor start-up funds (J.T.B.).

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Author notes

  1. Jason T. Boock

    Present address: Department of Chemical, Paper and Biomedical Engineering, Miami University (OH), Oxford, OH, USA

Authors and Affiliations

  1. Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA

    Jason T. Boock, Robert J. Conrado & Matthew P. DeLisa

  2. Department of Microbiology, Cornell University, Ithaca, NY, USA

    May Taw

  3. Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY, USA

    Brian C. King & Donna M. Gibson

  4. USDA Agricultural Research Service, Robert W. Holley Center for Agriculture and Health, Ithaca, NY, USA

    Donna M. Gibson

Authors
  1. Jason T. Boock
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  2. May Taw
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  3. Brian C. King
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  4. Robert J. Conrado
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  5. Donna M. Gibson
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  6. Matthew P. DeLisa
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Corresponding author

Correspondence to Jason T. Boock .

Editor information

Editors and Affiliations

  1. Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Caldes de Montbui, Spain

    Elena Garcia Fruitós

  2. Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Caldes de Montbui, Spain

    Anna Arís Giralt

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Boock, J.T., Taw, M., King, B.C., Conrado, R.J., Gibson, D.M., DeLisa, M.P. (2022). Two-Tiered Selection and Screening Strategy to Increase Functional Enzyme Production in E. coli. In: Garcia Fruitós, E., Arís Giralt, A. (eds) Insoluble Proteins. Methods in Molecular Biology, vol 2406. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1859-2_10

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  • DOI: https://doi.org/10.1007/978-1-0716-1859-2_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1858-5

  • Online ISBN: 978-1-0716-1859-2

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