When Second Best Is Good Enough: Another Probabilistic Look at Saturation Mutagenesis

We developed new criteria for determining the library size in a saturation mutagenesis experiment. When the number of all pos-sible distinct variants is large, any of the top-performing variants (e.g., any of the top three) is likely to meet the design require-ments, so the probability that the library contains at least one of them is a sensible criterion for determining the library size. By using a criterion of this type, one may significantly reduce the library size and thus save costs and labor while minimally compro-mising the quality of the best variant discovered. We present the probabilistic tools underlying these criteria and use them to compare the efficiencies of four randomization schemes: NNN, which uses all 64 codons; NNB, which uses 48 codons; NNK, which uses 32 codons; and MAX, which assigns equal probabilities to each of the 20 amino acids. MAX was found to be the most efficient randomization scheme and NNN the least efficient. TopLib, a computer program for carrying out the related calcula-tions, is available through a user-friendly Web server. S aturation mutagenesis (also called oligonucleotide-directed randomization) is a protein-engineering technique that has been used widely and successfully to improve protein properties such as catalytic activity, enantioselectivity, thermostability, and binding affinity (3, 12, 14, 16). We use the term “activity ” for th