Nondegenerate Saturation Mutagenesis: Library Construction and Analysis via MAX and ProxiMAX Randomization.
Amino acids
Codon randomization
Genetic code
Genetic diversity
Library design
Nondegenerate
Oligonucleotides
Protein engineering
Randomized gene libraries
Saturation mutagenesis
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
Protein engineering can enhance desirable features and improve performance outside of the natural context. Several strategies have been adopted over the years for gene diversification, and engineering of modular proteins in particular is most effective when a high-throughput, library-based approach is employed. Nondegenerate saturation mutagenesis plays a dynamic role in engineering proteins by targeting multiple codons to generate massively diverse gene libraries. Herein, we describe the nondegenerate saturation mutagenesis techniques that we have developed for contiguous (ProxiMAX) and noncontiguous (MAX) randomized codon generation to create precisely defined, diverse gene libraries, in the context of other fully nondegenerate strategies. ProxiMAX randomization comprises saturation cycling with repeated cycles of blunt-ended ligation, type IIS restriction, and PCR amplification, and is now a commercially automated process predominantly used for antibody library generation. MAX randomization encompasses a manual process of selective hybridisation between individual custom oligonucleotide mixes and a conventionally randomized template and is principally employed in the research laboratory setting, to engineer alpha helical proteins and active sites of enzymes. DNA libraries generated using either technology create high-throughput amino acid substitutions via codon randomization, to generate genetically diverse clones.
Identifiants
pubmed: 35727442
doi: 10.1007/978-1-0716-2152-3_3
doi:
Substances chimiques
Codon
0
Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
19-41Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/L015633/1
Pays : United Kingdom
Informations de copyright
© 2022. Springer Science+Business Media, LLC, part of Springer Nature.
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