Random insertion and deletion mutagenesis for construction of protein library containing nonnatural amino acids

A new method was developed for the generation of a library of mutant proteins that contained nonnatural amino acids. The method, "random insertion and deletion (RID) mutagenesis", is based on the deletion of an arbitrary number of bases at random positions and, at the same time, the insertion of an arbitrary sequence into the same position. By using this method, randomly selected three consecutive bases in the gene of green fluorescence protein (GFP) were replaced by a CGGT 4-base codon. When this DNA library was expressed in E. coli, about 80% of colonies lost the fluorescence. The non-fluorescent colonies were picked up and the genes were sequenced. Replacement of three consecutive bases by CGGT 4-base codon was found in two of the four mutated genes.