hide
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.
