title: Automated high-throughput process for site-directed mutagenesis, production, purification, and kinetic characterization of enzymes.
abstract: Site-directed mutagenesis followed by functional characterization is a widely used approach to obtain information on the structure-function relationship of proteins. Due to time and cost considerations, the number of amino acids studied is frequently reduced. To address the need for convenient parallel production of numerous point mutants of a protein, we developed an automated method to perform classical site-directed mutagenesis, protein purification, and characterization in a high-throughput manner. The process consists of a succession of six fully automated protocols that can be adapted to any automated liquid handling systems. Our procedure allows construction, validation, and characterization of hundreds of site-directed mutants of a given protein in just 4 days. The method is especially adapted to projects aiming at the study of unique or multiple mutants without the need to construct and screen large libraries of random mutants. The usefulness of the technique is illustrated by the construction and characterization of tens of single mutants of the penicillin-binding protein 2x (PBP2x) from Streptococcus pneumoniae. Moreover, seven mutations of PBP2x were obtained simultaneously in a single experiment with efficiency close to 90%.
authors.affiliation: Laboratoire d'Ingénierie des Macromolécules, Institut de Biologie Structurale (CEA/CNRS UMR 5075/UJF), Grenoble Cedex 1 F-38027, France.
authors.forename: Raphaël
authors.initials: R
authors.lastname: Carapito
authors.forename: Benoit
authors.initials: B
authors.lastname: Gallet
authors.forename: André
authors.initials: A
authors.lastname: Zapun
authors.forename: Thierry
authors.initials: T
authors.lastname: Vernet
mesh_terms: D004798:Enzymes / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D046915:Penicillin-Binding Proteins / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D004798:Enzymes; D050505:Mutant Proteins; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; C072223:PBP 2x protein, Streptococcus