Optimized and Automated Protocols for High-Throughput Screening of Amylosucrase Libraries
| Autor: | Magali Remaud-Simeon, Hakim Kharrat, Stéphane Emond, Pierre Monsan, Philippe Mondon, Khalil Bouayadi, Gabrielle Potocki-Veronese |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
DNA
Bacterial 0301 basic medicine Hot Temperature Time Factors High-throughput screening Genetic Vectors Drug Evaluation Preclinical Mutagenesis (molecular biology technique) Biology medicine.disease_cause Models Biological Sensitivity and Specificity 01 natural sciences Biochemistry Analytical Chemistry Automation 03 medical and health sciences Amylosucrase Transformation Genetic Enzyme Stability Escherichia coli medicine Dimethyl Sulfoxide Genomic library Selection Genetic Gene Library Thermostability Expression vector Genetic Variation Reproducibility of Results Water Directed evolution Molecular biology Recombinant Proteins 0104 chemical sciences 010404 medicinal & biomolecular chemistry 030104 developmental biology Genes Bacterial Glucosyltransferases Mutagenesis Site-Directed Solvents biology.protein Molecular Medicine Directed Molecular Evolution Biotechnology |
| Zdroj: | SLAS Discovery. 12:715-723 |
| ISSN: | 2472-5552 |
| Popis: | This article describes the design and validation of a general procedure for the high-throughput isolation of amylosucrase variants displaying higher thermostability or increased resistance to organic solvents. This procedure consists of 2 successive steps: an in vivo selection that eliminates inactive variants followed by automated screening of active variants to isolate mutants displaying enhanced features. The authors chose an Escherichia coli expression vector, allowing a high production rate of the recombinant enzyme in miniaturized culture conditions. The screening assay was validated by minimizing variability for various parameters of the protocol, especially bacterial growth and protein production in cultures in 96-well microplates. Recombinant amylosucrase production was normalized by decreasing the coefficient of variance from 27% to 12.5%. Selective screening conditions were defined to select variants displaying higher thermostability or increased resistance to organic solvents. A first-generation amylosucrase variant library, constructed by random mutagenesis, was subjected to this procedure, yielding a mutant displaying a 25-fold increased stability at 50 degrees C compared to the parental wild-type enzyme. |
| Databáze: | OpenAIRE |
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