ArticleNucleic acids research2004
In vitro selection of restriction endonucleases by in vitro compartmentalization.
Article in Nucleic acids research, 2004. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed, 50 citations in OpenAlex.
- Directed evolution ofReaction chemistry & engineering · 2023Article
- Ultrahigh-Throughput Enzyme Engineering and Discovery inChemical reviews · 2023Review
- Split & mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins.Nucleic acids research · 2020Article
- An in vitro compartmentalization-based method for the selection of bond-forming enzymes from large libraries.Biotechnology and bioengineering · 2016Article
- Expanding the scope of site-specific recombinases for genetic and metabolic engineering.Biotechnology and bioengineering · 2014Review
- In vitro evolution of enzymes.Methods in molecular biology (Clifton, N.J.) · 2013Article
- Cell-free synthetic biology: thinking outside the cell.Metabolic engineering · 2012Review
- An efficient method to assemble linear DNA templates for in vitro screening and selection systems.Nucleic acids research · 2009Article
- Applications of flow cytometry in environmental microbiology and biotechnology.Extremophiles : life under extreme conditions · 2009Review
- Interplay of metagenomics and in vitro compartmentalization.Microbial biotechnology · 2009Review
- Selection of restriction endonucleases using artificial cells.Nucleic acids research · 2007Article
- Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization.Proceedings of the National Academy of Sciences of the United States of America · 2005Article
- Laboratory-directed protein evolution.Microbiology and molecular biology reviews : MMBR · 2005Review
Corrections and comments
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.