title: A tripartite fusion system for the selection of protein variants with increased stability in vivo.
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.
authors: Howard Hughes Medical Institute, Chevy Chase, MD, USA.
authors: Linda
authors: L
authors: Foit
authors: James C A
authors: JC
authors: Bardwell
mesh_terms: D009154:Mutation; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics
chemical_list: D001618:beta-Lactamases