Fine-Tuning Enzyme Activity Through Saturation Mutagenesis

Codon saturation is a powerful tool for analyzing protein structure–function relationships and fine-tuning enzyme activity. In this technique, one or more key amino acids are randomized by incorporating degenerate codon(s) into a gene of interest in a polymerase-mediated primer extension reaction. Traditional methods for codon saturation mutagenesis are labor-intensive and typically require multiple rounds of PCR and restriction/ligation-based cloning. In contrast, the QuikChange^� Multi Site-Directed Mutagenesis kit provides an efficient one-day procedure for incorporating degenerate codons into any double-stranded plasmid DNA template. Originally developed for introducing point mutations at multiple sites, the Multi kit can be readily adapted for performing site-directed saturation mutagenesis of one, two, or three amino acids simultaneously. When coupled with a suitable screening assay, this method simplifies the process of surveying multiple side chain replacements at key locations.