ArticleNucleic acids research2008
Linking the functions of unrelated proteins using a novel directed evolution domain insertion method.
Article in Nucleic acids research, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 43 papers.
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Who cites it
43 citing papers in PubMed, 93 citations in OpenAlex.
- Fusion Protein Technology to Enhance Pharmacological Properties of L-Asparaginases.Biomolecules · 2026Review
- How far can you go? Extrapolating values of catalytic activity from known protein landscapes in natural and directed evolution.Chemical Society reviews · 2026Review
- High-throughput methods enabling random duplications, deletions, or nucleotide-constrained mutagenesis of entire DNA motifs.Nucleic acids research · 2026Article
- A Novel Biosensor for Ferrous Iron Developed via CoBiSe: A Computational Method for Rapid Biosensor Design.ACS sensors · 2026Article
- Parallel laboratory evolution and rational debugging reveal genomic plasticity toCell genomics · 2023Article
- Dissecting the Determinants of Domain Insertion Tolerance and Allostery in Proteins.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2023Article
- DIMPLE: deep insertion, deletion, and missense mutation libraries for exploring protein variation in evolution, disease, and biology.Genome biology · 2023Article
- Designed Artificial Protein Heterodimers With Coupled Functions Constructed Using Bio-Orthogonal Chemistry.Frontiers in chemistry · 2021Article
- Design of Protein Logic Gate System Operating on Lipid Membranes.ACS synthetic biology · 2020Article
- Targeted insertional mutagenesis libraries for deep domain insertion profiling.Nucleic acids research · 2020Article
- Genetically Engineered Proteins to Improve Biomass Conversion: New Advances and Challenges for Tailoring Biocatalysts.Molecules (Basel, Switzerland) · 2019Review
- Engineering proteins for allosteric control by light or ligands.Nature protocols · 2019Article
- Development and structural characterization of an engineered multi-copper oxidase reporter of protein-protein interactions.The Journal of biological chemistry · 2019Article
- Domain insertion permissibility-guided engineering of allostery in ion channels.Nature communications · 2019Article
- Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion.BioMed research international · 2019Review
- Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering.Nucleic acids research · 2018Article
- Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion.Protein engineering, design & selection : PEDS · 2018Article
- Rapid construction of metabolite biosensors using domain-insertion profiling.Nature communications · 2016Article
- Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.Nature biotechnology · 2016Article
- Transposon for protein engineering.Mobile genetic elements · 2016Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
We have successfully developed a new directed evolution method for generating integral protein fusions comprising of one domain inserted within another. Creating two connections between the insert and accepting parent domain can result in the inter-dependence of the separate protein activities, thus providing a general strategy for constructing molecular switches. Using an engineered transposon termed MuDel, contiguous trinucleotide sequences were removed at random positions from the bla gene encoding TEM-1 beta-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b(562) with differing linking sequences at each terminus and sampling all three reading frames. The result was a variety of chimeric genes encoding novel integral fusion proteins that retained TEM-1 activity. While most of the tolerated insertions were observed in loops, several also occurred close to the termini of alpha-helices and beta-strands. Several variants conferred a switching phenotype on Escherichia coli, with bacterial tolerance to ampicillin being dependent on the presence of haem in the growth medium. The magnitude of the switching phenotype ranged from 4- to 128-fold depending on the insertion position within TEM-1 and the linker sequences that join the two domains.
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Registered trials
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.