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  Reducing Codon Redundancy and Screening Effort of Combinatorial Protein Libraries Created by Saturation Mutagenesis

Kille, S., Acevedo-Rocha, C. G., Parra, L. P., Zhang, Z.-G., Oppermann, D. J., Reetz, M. T., et al. (2013). Reducing Codon Redundancy and Screening Effort of Combinatorial Protein Libraries Created by Saturation Mutagenesis. ACS Synthetic Biology, (2), 83-92. doi:10.1021/sb300037w.

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 Creators:
Kille, Sabrina1, 2, Author              
Acevedo-Rocha, Carlos G.1, 2, Author              
Parra, Loreto P.1, 2, Author              
Zhang, Zhi-Gang1, 2, Author              
Oppermann, Diederik J.1, 2, Author              
Reetz, Manfred T.1, 2, Author              
Acevedo, Juan Pablo3, Author              
Affiliations:
1Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, Kaiser-Wilhelm-Platz 1, 45470 Mülheim, DE, ou_1445588              
2Fachbereich Chemie, Philipps-Universität Marburg, , Hans-Meerwein-Str., 35032 Marburg, DE, ou_persistent22              
3Facultad de Medicina y Facultad de Ingenieria de la Univerisidad de los Andes, Santiago, Chile, ou_persistent22              

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Free keywords: codon redundancy primer degeneracy screening effort library quality directed evolution combinatorial mutagenesis
 Abstract: Saturation mutagenesis probes define sections of the vast protein sequence space. However, even if randomization is limited this way, the combinatorial numbers problem is severe. Because diversity is created at the codon level, codon redundancy is a crucial factor determining the necessary effort for library screening. Additionally, due to the probabilistic nature of the sampling process, oversampling is required to ensure library completeness as well as a high probability to encounter all unique variants. Our trick employs a special mixture of three primers, creating a degeneracy of 22 unique codons coding for the 20 canonical amino acids. Therefore, codon redundancy and subsequent screening effort is significantly reduced, and a balanced distribution of codon per amino acid is achieved, as demonstrated exemplarily for a library of cyclohexanone monooxygenase. We show that this strategy is suitable for any saturation mutagenesis methodology to generate less-redundant libraries.

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Language(s): eng - English
 Dates: 2012-06-152013-02-15
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/sb300037w
 Degree: -

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Title: ACS Synthetic Biology
Source Genre: Journal
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Pages: - Volume / Issue: (2) Sequence Number: - Start / End Page: 83 - 92 Identifier: ISSN: 2161-5063