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

 Last Update: 2021-01-05
 Source: Internet
 Author: User

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antibiotic resistance win

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

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