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A Tripartite Fusion System for the Selection of Protein Variants with Increased Stability In Vivo

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Enzyme Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 978))

Abstract

We describe here a genetic selection system that directly links protein stability to antibiotic resistance, allowing one to directly select for mutations that stabilize proteins in vivo. Our technique is based on a tripartite fusion in which the protein to be stabilized is inserted into the middle of the reporter protein β-lactamase via a flexible linker. The gene encoding the inserted protein is then mutagenized using error-prone PCR and the resulting plasmid library plated on media supplemented with increasing concentrations of β-lactam antibiotic. Mutations that stabilize the protein of interest can easily be identified on the basis of their increased antibiotic resistance compared to cells expressing the unmutated tripartite fusion.

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Author information

Authors and Affiliations

  1. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    Linda Foit & James C. A. Bardwell

  2. Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Linda Foit & James C. A. Bardwell

Authors
  1. Linda Foit
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  2. James C. A. Bardwell
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Corresponding author

Correspondence to James C. A. Bardwell .

Editor information

Editors and Affiliations

  1. New England Biolabs, Inc., County Road 240, Ipswich, 01938, Massachusetts, USA

    James C. Samuelson

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© 2013 Springer New York

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Foit, L., Bardwell, J.C.A. (2013). A Tripartite Fusion System for the Selection of Protein Variants with Increased Stability In Vivo. In: Samuelson, J. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 978. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-293-3_1

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  • DOI: https://doi.org/10.1007/978-1-62703-293-3_1

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-292-6

  • Online ISBN: 978-1-62703-293-3

  • eBook Packages: Springer Protocols

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