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

Cassette mutagenesis is a method of protein engineering which generates a wide diversity of genetic variants that can be subjected to either selection or screening. As long as the target sequence to be modified is kept short (corresponding to four to six amino acids), complete combinatorial libraries can be produced. A major problem arises when longer peptides are to be engineered for desired functions. In such situations the production of a limited collection of variants can be helpful; thus, biased random mutagenesis and 'doping schemes' have been reported previously. Here we describe a computer algorithm that enables the determination of the degree of phosphoramidite contamination of nucleotide precursor reservoirs. Through simulation of biological translation, the algorithm allows the prediction of the effect of contamination levels on the number of mutations to occur for any given peptide sequence. In this study the cholinergic binding site was used as a model sequence (22 amino acids). Considerations, based on the computer program, are discussed regarding the efficient design of phage-display combinatorial libraries.