ArticleProceedings of the National Academy of Sciences of the United States of America2005
Evolution of highly active enzymes by homology-independent recombination.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2005. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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Who cites it
18 citing papers in PubMed, 56 citations in OpenAlex.
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- Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening.Scientific reports · 2016Article
- Increased antibody affinity confers broad in vitro protection against escape mutants of severe acute respiratory syndrome coronavirus.Journal of virology · 2012Article
- Structure-based design of supercharged, highly thermoresistant antibodies.Chemistry & biology · 2012Article
- Assessing directed evolution methods for the generation of biosynthetic enzymes with potential in drug biosynthesis.Future medicinal chemistry · 2011Article
- Prospecting metagenomic enzyme subfamily genes for DNA family shuffling by a novel PCR-based approach.The Journal of biological chemistry · 2010Article
- Physiological roles of glutathione s-transferases in soybean root nodules.Plant physiology · 2009Article
- Engineering antibody fragments to fold in the absence of disulfide bonds.Protein science : a publication of the Protein Society · 2009Article
- Construction and flow cytometric screening of targeted enzyme libraries.Nature protocols · 2009Article
- Recombination of protein fragments: a promising approach toward engineering proteins with novel nanomechanical properties.Protein science : a publication of the Protein Society · 2008Article
- Synthetic antibody libraries focused towards peptide ligands.Journal of molecular biology · 2008Article
- Revealing biases inherent in recombination protocols.BMC biotechnology · 2007Article
- Non-homologous recombination of deoxyribonucleoside kinases from human and Drosophila melanogaster yields human-like enzymes with novel activities.Journal of molecular biology · 2007Article
- APEx 2-hybrid, a quantitative protein-protein interaction assay for antibody discovery and engineering.Proceedings of the National Academy of Sciences of the United States of America · 2007Article
- Diversification of catalytic function in a synthetic family of chimeric cytochrome p450s.Chemistry & biology · 2007Article
- The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase.Journal of molecular biology · 2006Article
- IPRO: an iterative computational protein library redesign and optimization procedure.Biophysical journal · 2006Article
Corrections and comments
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Authors and funding
6 authors at 2 institutions in 2 countries.
Funding
Abstract
The theta-class GST enzymes hGSTT1-1 (human GSTTheta-1-1) and rGSTT2-2 (rat GSTTheta-2-2) share 54.3% amino acid identity and exhibit different substrate specificities. Homology-independent techniques [incremental truncation for the creation of hybrid enzymes (ITCHY) and SCRATCHY] and low-homology techniques (recombination-dependent exponential amplification PCR) were used to create libraries of chimeric enzymes containing crossovers (C/Os) at positions not accessible by DNA family shuffling. High-throughput flow cytometric screening using the fluorogenic rGSTT2-2-specific substrate 7-amino-4-chloromethyl coumarin led to the isolation of active variants with either one or two C/Os. One of these enzymes, SCR23 (83% identity to hGSTT1-1), was encoded by a gene that exchanged helices 4 and 5 of hGSTT1-1 with the corresponding sequence from rGSTT2-2. Compared with either parent, this variant was found to have an improved k(cat) with the selection substrate and also exhibited activity for the conjugation of glutathione to ethacrynic acid, a compound that is not recognized by either parental enzyme. These results highlight the power of combinatorial homology-independent and low-homology recombination methods for the generation of unique, highly active enzymes and also suggest a possible means of enzyme "humanization."
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