ArticleProceedings of the National Academy of Sciences of the United States of America2004
Directed evolution of protein enzymes using nonhomologous random recombination.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2004. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed, 59 citations in OpenAlex.
- Modern methods for laboratory diversification of biomolecules.Current opinion in chemical biology · 2017Review
- Methods for the directed evolution of proteins.Nature reviews. Genetics · 2015Review
- Domain-swapping of mesophilic xylanase with hyper-thermophilic glucanase.BMC biotechnology · 2012Article
- Prospecting metagenomic enzyme subfamily genes for DNA family shuffling by a novel PCR-based approach.The Journal of biological chemistry · 2010Article
- Design, selection, and characterization of a split chorismate mutase.Protein science : a publication of the Protein Society · 2010Article
- Re-engineering a beta-lactamase using prototype peptides from a library of local structural motifs.Protein science : a publication of the Protein Society · 2009Article
- Recombination of protein fragments: a promising approach toward engineering proteins with novel nanomechanical properties.Protein science : a publication of the Protein Society · 2008Article
- Selecting folded proteins from a library of secondary structural elements.Journal of the American Chemical Society · 2008Article
- Motif programming: a microgene-based method for creating synthetic proteins containing multiple functional motifs.Nucleic acids research · 2007Article
- Discovery of a mRNA mitochondrial localization element in Saccharomyces cerevisiae by nonhomologous random recombination and in vivo selection.Nucleic acids research · 2007Article
- Identification of eukaryotic promoter regulatory elements using nonhomologous random recombination.Nucleic acids research · 2007Article
- Laboratory-directed protein evolution.Microbiology and molecular biology reviews : MMBR · 2005Review
- Simultaneous optimization of enzyme activity and quaternary structure by directed evolution.Protein science : a publication of the Protein Society · 2005Article
- Evolution of highly active enzymes by homology-independent recombination.Proceedings of the National Academy of Sciences of the United States of America · 2005Article
- Ribozyme motif structure mapped using random recombination and selection.RNA (New York, N.Y.) · 2005Article
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
We recently reported the development of nonhomologous random recombination (NRR) as a method for nucleic acid diversification and applied NRR to the evolution of DNA aptamers. Here, we describe a modified method, protein NRR, that enables proteins to access diversity previously difficult or impossible to generate. We investigated the structural plasticity of protein folds and the ability of helical motifs to function in different contexts by applying protein NRR and in vivo selection to the evolution of chorismate mutase (CM) enzymes. Functional CM mutants evolved using protein NRR contained many insertions, deletions, and rearrangements. The distribution of these changes was not random but clustered in certain regions of the protein. Topologically rearranged but functional enzymes also emerged from these studies, indicating that multiple connectivities can accommodate a functional CM active site and demonstrating the ability to generate new domain connectivities through protein NRR. Protein NRR was also used to randomly recombine CM and fumarase, an unrelated but also alpha-helical protein. Whereas the resulting library contained fumarase fragments in many contexts before functional selection, library members surviving selection for CM activity invariably contained a CM core with fumarase sequences found only at the termini or in one loop. These results imply that internal helical fragments cannot be swapped between these proteins without the loss of nearly all CM activity. Our findings suggest that protein NRR will be useful in probing the functional requirements of enzymes and in the creation of new protein topologies.
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