CODON-BASED COMBINATORIAL ALANINE SCANNING SITE-DIRECTED MUTAGENESIS - DESIGN, IMPLEMENTATION, AND POLYMERASE CHAIN-REACTION SCREENING

Combinatorial alanine scanning mutagenesis is a powerful tool for the exploration of protein structure-function relationships. Unfortunately, combinatorial alanine replacement of multiple residues using standard site-directed mutagenesis is restricted to a subset of amino acids. To circumvent this limitation, an efficient-procedure for combinatorial site-specific replacement by alanine of any residue in a given protein sequence has been established. The method, which involves simple procedures and commonly used materials, is based upon the use of codon-based mutagenesis. A defined ratio of alanine to wild-type codon was introduced at each predetermined triplet using the ''column-splitting'' technique during oligonucleotide synthesis. High-throughput genetic screening of mutant libraries was facilitated by the incorporation of diagnostic restriction sites at targeted codons followed by a PCR-based screening procedure. The method was tested on a set of 13 residues located at the interface between the variable domains of a Fab fragment-of an antibody. The occurrence of alanine substitutions was found to be comparable to the statistically predicted distribution. (C) 1995 Academic Press, Inc.