BIASED RANDOM MUTAGENESIS OF PEPTIDES - DETERMINATION OF MUTATION FREQUENCY BY COMPUTER-SIMULATION

Cassette mutagenesis is a method of protein engineering which generate s 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 co mbinatorial libraries can be produced. A major problem arises when lon ger peptides are to be engineered for desired functions. In such situa tions the production of a limited collection of variants can be helpfu l; thus, biased random mutagenesis and 'doping schemes' have been repo rted previously. Here we describe a computer algorithm that enables th e determination of the degree of phosphoramidite contamination of nucl eotide precursor reservoirs. Through simulation of biological translat ion, the algorithm allows the prediction of the effect of contaminatio n levels on the number of mutations to occur for any given peptide seq uence. In this study the cholinergic binding site was used as a model sequence (22 amino acids). Considerations, based on the computer progr am, are discussed regarding the efficient design of phage-display comb inatorial libraries.