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Enzyme Engineering

Volume 978 of the series Methods in Molecular Biology pp 1-20

Date:

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

  • Linda FoitAffiliated withHoward Hughes Medical InstituteDepartment of Molecular, Cellular and Developmental Biology, University of Michigan
  • , James C. A. BardwellAffiliated withHoward Hughes Medical InstituteDepartment of Molecular, Cellular and Developmental Biology, University of Michigan Email author 

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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.

Key words

Genetic selection Protein stability Protein evolution Mutagenesis Reporter protein Tripartite fusion Sandwich fusion