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

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons58686

Kille,  Sabrina
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Fachbereich Chemie, Philipps-Universität Marburg, ;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons132851

Acevedo-Rocha,  Carlos G.
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Fachbereich Chemie, Philipps-Universität Marburg, ;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons58869

Parra,  Loreto P.
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Fachbereich Chemie, Philipps-Universität Marburg, ;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons59138

Zhang,  Zhi-Gang
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Fachbereich Chemie, Philipps-Universität Marburg, ;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons132998

Oppermann,  Diederik J.
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Fachbereich Chemie, Philipps-Universität Marburg, ;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons58919

Reetz,  Manfred T.
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Fachbereich Chemie, Philipps-Universität Marburg, ;

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Zitation

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.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0010-5129-F
Zusammenfassung
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.