ArticlePloS one2014
Site-specific bioconjugation of a murine dihydrofolate reductase enzyme by copper(I)-catalyzed azide-alkyne cycloaddition with retained activity.
Article in PloS one, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
What it found
Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.
The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.
Who cites it
14 citing papers in PubMed, 40 citations in OpenAlex.
- Noncanonical Amino Acids in Biocatalysis.Chemical reviews · 2024Review
- Employing non-canonical amino acids towards the immobilization of a hyperthermophilic enzyme to increase protein stability.RSC advances · 2023Article
- Non-canonical amino acids as a tool for the thermal stabilization of enzymes.Protein engineering, design & selection : PEDS · 2023Review
- Engineering of enzymes using non-natural amino acids.Bioscience reports · 2022Review
- Folding Assessment of Incorporation of Noncanonical Amino Acids Facilitates Expansion of Functional-Group Diversity for Enzyme Engineering.Chemistry (Weinheim an der Bergstrasse, Germany) · 2020Article
- Playing with the Molecules of Life.ACS chemical biology · 2018Review
- Practical Considerations, Challenges, and Limitations of Bioconjugation via Azide-Alkyne Cycloaddition.Bioconjugate chemistry · 2018Review
- Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex.Scientific reports · 2016Article
- Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein.Scientific reports · 2016Article
- Expanding the scope of alkyne-mediated bioconjugations utilizing unnatural amino acids.Chemical communications (Cambridge, England) · 2016Article
- Click chemistry for targeted protein ubiquitylation and ubiquitin chain formation.Nature protocols · 2015Article
- Effects of Non-Natural Amino Acid Incorporation into the Enzyme Core Region on Enzyme Structure and Function.International journal of molecular sciences · 2015Article
- Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo.Journal of controlled release : official journal of the Controlled Release Society · 2015Article
- Site-specific bioconjugation of an organometallic electron mediator to an enzyme with retained photocatalytic cofactor regenerating capacity and enzymatic activity.Molecules (Basel, Switzerland) · 2015Article
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
5 authors at 4 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA) containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR), in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activity.
Indexed as
Identifiers
What OpenQuestion holds
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.