Extracting information from protein sequences using random replacement mutagenesis

This paper describes a mutagenesis technique that involves randomizing the DNA sequence of a short stretch of a gene using a synthetic linker with the recognition sequence of a type IIs restriction endonuclease at each end. Type IIs endonucleases cleave DNA at precise distances from their recognition sequences. Linker insertion mutagenesis was used to generate a collection of individual linker inserts distributed throughout the target gene. Then, the type IIs enzyme was used to release the linker and create a precise deletion in the gene. A second linker containing random nucleotide sequence was ligated to the gene to replace the deleted DNA with random sequence DNA. The result was a plasmid library containing all or nearly all possible substitutions for a given region of a protein. We applied this method to the gene encoding TEM-1 β-lactamase and have illustrated that the mutagenesis method is an effective strategy for defining regions important to the structure and function of a protein. Methods for troubleshooting and improving the mutagenesis technique are also described.