Biased random mutagenesis of peptides: determination of mutation frequency by computer simulation

Cassette mutagenesis is a method of protein engineering whichgenerates a wide diversity of genetic variants that can be subjectedto either selection or screening. As long as the target sequenceto be modified is kept short (corresponding to four to six aminoacids), complete combinatorial libraries can be produced. Amajor problem arises when longer peptides are to be engineeredfor desired functions. In such situations the production ofa limited collection of variants can be helpful; thus, biasedrandom mutagenesis and ‘doping schemes’ have beenreported previously. Here we describe a computer algorithm thatenables the determination of the degree of phosphoramidite contaminationof nucleotide precursor reservoirs. Through simulation of biologicaltranslation, the algorithm allows the prediction of the effectof contamination levels on the number of mutations to occurfor any given peptide sequence. In this study the cholinergicbinding site was used as a model sequence (22 amino acids).Considerations, based on the computer program, are discussedregarding the efficient design of phage-display combinatoriallibraries.