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Nucleic Acids Symposium Series 2000 44(1):69-70; doi:10.1093/nass/44.1.69
© 2000 by Oxford University Press
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Random insertion and deletion mutagenesis for construction of protein library containing nonnatural amino acids

Hiroshi Murakami, Takahiro Hohsaka and Masahiko Sisido

Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan

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-basecodon. 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.


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