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Site-saturation mutagenesis (SSM) has been used in directed evolution of proteins for a long time. As a special form of saturation mutagenesis, it involves individual randomization at a given residue with formation of all 19 amino acids. To date, the most efficient embodiment of SSM is a one-step PCR-based approach using NNK codon degeneracy. However, in the case of difficult-to-randomize genes, SSM may not deliver all of the expected 19 mutants, which compels the user to invest further efforts by applying site-directed mutagenesis for the construction of the missing mutants. To solve this problem, we developed a two-step PCR-based technique in which a mutagenic primer and a non-mutagenic (silent) primer are used to generate a short DNA fragment, which is recovered and then employed as a megaprimer to amplify the whole plasmid. The present two-step and older one-step (partially overlapped primer approach) procedures were compared by utilizing cytochrome P450-BM3, which is a "difficult-to-randomize" gene. The results document the distinct superiority of the new method by checking the library quality on DNA level based on massive sequence data, but also at amino acid level. Various future applications in biotechnology can be expected, including the utilization when constructing mutability landscapes, which provide semi-rational information for identifying hot spots for protein engineering and directed evolution.

Boosting the efficiency of site-saturation mutagenesis for a difficult-to-randomize gene by a two-step PCR strategy