title: Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation 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.
authors: Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany.
authors: Sabrina
authors: S
authors: Kille
authors: Carlos G
authors: CG
authors: Acevedo-Rocha
authors: Loreto P
authors: LP
authors: Parra
authors: Zhi-Gang
authors: ZG
authors: Zhang
authors: Diederik J
authors: DJ
authors: Opperman
authors: Manfred T
authors: MT
authors: Reetz
authors: Juan Pablo
authors: JP
authors: Acevedo
mesh_terms: D000596:Amino Acids / Q000235:genetics; D003062:Codon; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D015723:Gene Library; D005815:Genetic Code; D016254:Mutagenesis, Insertional / Q000379:methods*; D010105:Oxygenases / Q000235:genetics; D011506:Proteins / Q000235:genetics*
chemical_list: D000596:Amino Acids; D003062:Codon; D017931:DNA Primers; D011506:Proteins; D010105:Oxygenases; C019961:cyclohexanone oxygenase