Article thumbnail
Location of Repository

In vitro selection of restriction endonucleases by in vitro compartmentalization

By Nobuhide Doi, Shin Kumadaki, Yuko Oishi, Nobutaka Matsumura and Hiroshi Yanagawa

Abstract

Restriction endonucleases are widely used in laboratory applications from recombinant DNA technology to diagnostics, but engineering of restriction enzymes by structure-guided design and in vivo directed evolution is at an early stage. Here, we report the use of an in vitro compartmentalization system for completely in vitro selection of restriction enzymes. Compartmentalization of a single gene in a rabbit reticulocyte in vitro transcription/translation system serves to isolate individually synthesized enzymes from each other. In each compartment, an active enzyme cleaves only its own encoding gene, whereas genes encoding inactive enzymes remain intact. Affinity selection of the cleaved DNA encoding active restriction endonucleases was accomplished by the use of streptavidin-immobilized beads and dUTP-biotin, which was efficiently incorporated into the cohesive end of the cleaved DNA using a DNA polymerase. We confirmed that genes encoding active restriction endonuclease FokI could be selected from a randomized library. This method overcomes the limitations of current in vivo technologies and should prove useful for rapid screening and evolution of novel restriction enzymes from diverse mutant libraries, as well as for studies of catalytic and evolutionary mechanisms of restriction enzymes

Topics: NAR Methods Online
Publisher: Oxford University Press
Year: 2004
DOI identifier: 10.1093/nar/gnh096
OAI identifier: oai:pubmedcentral.nih.gov:484195
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://dx.doi.org/10.1093/nar/... (external link)
  • Suggested articles


    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.