Gene Site Saturation Mutagenesis: A Comprehensive Mutagenesis Approach

Abstract

This chapter describes the various aspects of gene site saturation mutagenesis (GSSM). GSSM systematically explores minimally all possible single amino acid substitutions along a protein sequence. This comprehensive technique introduces point mutations into every position within a target gene using degenerate primer sets containing 32 or 64 codons to generate a complete library of variants. It is found that unlike rational mutagenesis, GSSM does not require prior knowledge of the structure, or mechanism of the target protein due to its ability to generate all mutations at all positions within the protein. GSSM has been used to improve the thermostability of a haloalkane dehalogenase by 30,000-fold. It is found that when the mutations were analyzed at the eight single sites that individually improved thermostability, three of the substitutions would likely never have been accessed by routine procedures, such as error-prone polymerase chain reaction independent of sampling scale. Single nucleotide substitutions result in a theoretical maximum of 5–6 amino acid changes per codon with only 2–3 amino acid substitutions generally accessed with standard protocols. It is found that GSSM enables a complete analysis of every position in a given gene.