In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent SpecificityKrzysztof J. Skowronek 1, Matthias Bochtler 1
1International Institute of Molecular and Cell Biology, Warsaw (IIMCB)
Restriction endonucleases with new sequence specificity can be developed from enzymes recognizing a partially degenerate sequence. Here we provide a detailed protocol that we successfully used to alter the sequence specificity of NlaIV enzyme. Key ingredients of the protocol are the in vitro compartmentalization of the transcription/translation reaction and selection of variants with new sequence specificities.
