Nondegenerate Saturation Mutagenesis: Library Construction and Analysis via MAX and ProxiMAX Randomization

Anupama Chembath, Ben P G Wagstaffe, Mohammed Ashraf, Marta M Ferreira Amaral, Laura Frigotto, Anna V Hine*

*Corresponding author for this work

Research output: Chapter in Book/Published conference outputChapter (peer-reviewed)peer-review


Protein engineering can enhance desirable features and improve performance outside of the natural context. Several strategies have been adopted over the years for gene diversification, and engineering of modular proteins in particular is most effective when a high-throughput, library-based approach is employed. Nondegenerate saturation mutagenesis plays a dynamic role in engineering proteins by targeting multiple codons to generate massively diverse gene libraries. Herein, we describe the nondegenerate saturation mutagenesis techniques that we have developed for contiguous (ProxiMAX) and noncontiguous (MAX) randomized codon generation to create precisely defined, diverse gene libraries, in the context of other fully nondegenerate strategies. ProxiMAX randomization comprises saturation cycling with repeated cycles of blunt-ended ligation, type IIS restriction, and PCR amplification, and is now a commercially automated process predominantly used for antibody library generation. MAX randomization encompasses a manual process of selective hybridisation between individual custom oligonucleotide mixes and a conventionally randomized template and is principally employed in the research laboratory setting, to engineer alpha helical proteins and active sites of enzymes. DNA libraries generated using either technology create high-throughput amino acid substitutions via codon randomization, to generate genetically diverse clones.

Original languageEnglish
Title of host publicationDirected Evolution
Subtitle of host publication Methods in Molecular Biology
EditorsA. Currin, N. Swainston
Number of pages23
ISBN (Electronic)978-1-0716-2152-3
ISBN (Print)978-1-0716-2151-6
Publication statusPublished - 22 Jun 2022

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Bibliographical note

Funding Information:
This project has received funding from the European Union Horizon 2020 FET OPEN program under grant agreement No. 764434 (A.C., B.P.G.W., and M.A.) and BBSRC Grant No. BB/L015633/1 (M.M.F.A.).


  • Protein engineering
  • Nondegenerate
  • Saturation mutagenesis
  • Codon randomization
  • Mutagenesis
  • Amino acids
  • Codon - genetics
  • Randomized gene libraries
  • Genetic diversity
  • Proteins - chemistry
  • Library design
  • Random Allocation
  • Oligonucleotides
  • Gene Library
  • Protein Engineering - methods
  • Genetic code


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