A Directed Evolution System for Lysine Deacetylases.
Methods Mol Biol
; 2247: 319-337, 2021.
Article
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| MEDLINE
| ID: mdl-33301126
Lysine acetylation is a ubiquitous modification permeating the proteomes of organisms from all domains of life. Lysine deacetylases (KDACs) reverse this modification by following two fundamentally different enzymatic mechanisms, which differ mainly by the need for NAD+ as stoichiometric co-substrate. KDACs are often found as catalytic subunit in protein complexes involved in cell cycle regulation, chromatin organization and transcription. Their promiscuity with respect to sequence context and type of lysine acylation convolutes the network of functional and physical connections.Here we present an efficient selection method for KDACs in E. coli, which allows for the creation of acyl-type specific KDAC variants, which greatly facilitate the investigation of their physiological function . The selection system builds on the incorporation of acylated lysines by genetic code expansion in reporter enzymes with essential lysine residues. We describe the creation of KDAC mutant libraries by saturation mutagenesis of active site residues, the isolation of individual mutants from this library using the selection system, and their biochemical characterization with acylated firefly luciferase.
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Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Evolución Biológica
/
Histona Desacetilasas
/
Lisina
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2021
Tipo del documento:
Article
País de afiliación:
Alemania
Pais de publicación:
Estados Unidos