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

详细信息    查看全文

• 作者：Sabrina Kille ; Carlos G. Acevedo-Rocha ; Loreto P. Parra ; Zhi-Gang Zhang ; Diederik J. Opperman ; Manfred T. Reetz ; Juan Pablo Acevedo
• 刊名：ACS Synthetic Biology
• 出版年：2013
• 出版时间：February 15, 2013
• 年：2013
• 卷：2
• 期：2
• 页码：83-92
• 全文大小：444K
• 年卷期：v.2,no.2(February 15, 2013)
• ISSN：2161-5063

文摘

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.

Keywords:

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