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-throughout 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 substitution was found to be comparable to the statistically predicted distribution.
D000409:Alanine; D001483:Base Sequence; D003062:Codon; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D013379:Substrate Specificity
D003062:Codon; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011506:Proteins; D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes; D000409:Alanine