Facile Assembly of Combinatorial Mutagenesis Libraries Using Nicking Mutagenesis.
Antibody engineering
Combinatorial mutagenesis
Directed evolution
Enzyme engineering
Epistasis
Fitness landscapes
Molecular evolution
Nicking mutagenesis
Protein engineering
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
21
6
2022
pubmed:
22
6
2022
medline:
24
6
2022
Statut:
ppublish
Résumé
Combinatorial mutagenesis is a method where multiple user-defined mutations are encoded at defined positions in a sequence. Combinatorial mutagenic libraries can be used in a variety of applications including evaluating fundamental questions about molecular evolution, directed evolution workflows for enzyme engineering, and in better understanding of biological processes like antibody affinity maturation. Here, we show a method of combinatorial mutagenesis utilizing the template-based nicking mutagenesis with several modifications. We show an example for generating a combinatorial library with 14 mutated positions, a total of 16,384 library variants, and a protocol for the generation of large, user-defined combinatorial libraries. The reader can use this protocol to create such libraries in 2 days.
Identifiants
pubmed: 35727445
doi: 10.1007/978-1-0716-2152-3_6
pmc: PMC9730894
mid: NIHMS1851581
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
85-109Subventions
Organisme : NIAID NIH HHS
ID : R01 AI141452
Pays : United States
Informations de copyright
© 2022. Springer Science+Business Media, LLC, part of Springer Nature.
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