Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis

Sabrina Kille, Carlos G. Acevedo-Rocha, Loreto P. Parra, Zhi Gang Zhang, Diederik J. Opperman, Manfred T. Reetz, Juan Pablo Acevedo

Research output: Contribution to journalArticlepeer-review

131 Scopus citations

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.
Original languageAmerican English
Pages (from-to)83-92
Number of pages10
JournalACS Synthetic Biology
Volume2
Issue number2
DOIs
StatePublished - 15 Feb 2013

Keywords

  • codon redundancy
  • combinatorial mutagenesis
  • directed evolution
  • library quality
  • primer degeneracy
  • screening effort

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