ArticleProtein science : a publication of the Protein Society2022
High-efficiency recombinant protein purification using mCherry and YFP nanobody affinity matrices.
Article in Protein science : a publication of the Protein Society, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 23 papers.
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Who cites it
23 citing papers in PubMed, 27 citations in OpenAlex.
- Molecular basis of allosteric regulation and pharmaceutical targeting of protein kinase Cβ.Nature communications · 2026Article
- Synthetic multicolor antigen-stabilizable nanobody platform for intersectional labeling and functional imaging.Nature methods · 2026Article
- Nanobodies in biomedicine: from molecular characteristics to fabrication and clinical translation.Military Medical Research · 2026Review
- Engineering a new tripartite split-ccGFP system from Corynactis californica for detecting protein-protein interactions.Scientific reports · 2025Article
- Synthetic multicolor antigen-stabilizable nanobody platform for intersectional labelling and functional imaging.bioRxiv : the preprint server for biology · 2025Article
- Integrative biophysical characterization of molecular interactions: A case study with the sfGFP-nanobody complex.Analytical biochemistry · 2025Article
- Topobexin targets the Topoisomerase II ATPase domain for beta isoform-selective inhibition and anthracycline cardioprotection.Nature communications · 2025Article
- Nanobodies: From Discovery to AI-Driven Design.Biology · 2025Review
- Unique mechanisms to increase structural stability and enhance antigen binding in nanobodies.Structure (London, England : 1993) · 2025Article
- PEG-mCherry interactions beyond classical macromolecular crowding.Protein science : a publication of the Protein Society · 2025Article
- A modular cell-free protein biosensor platform using split T7 RNA polymerase.Science advances · 2025Article
- Recombinant Topoisomerase 2 Production Using Cultured Human Cell Lines.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Topoisomerase II DNA Cleavage Assay Using Fluorescently Labelled Double-Stranded Oligonucleotides.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Yeast Tools for Studying Type II Topoisomerases in Budding Yeast.Methods in molecular biology (Clifton, N.J.) · 2025Article
- A new strategy to generate nanobodies for the coumaphos based on the synthesized nanobody libraries.Food chemistry · 2024Article
- Destabilized near-infrared fluorescent nanobodies enable background-free targeting of GFP-based biosensors for imaging and manipulation.Nature communications · 2024Article
- A modular cell-free protein biosensor platform using split T7 RNA polymerase.bioRxiv : the preprint server for biology · 2024Article
- Enzymatic Protein Immobilization for Nanobody Array.Molecules (Basel, Switzerland) · 2024Article
- ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes.Nature communications · 2023Article
- Production and Purification of Cysteine-Rich Leptospiral Virulence-Modifying Proteins with or Without mCherry Fusion.The protein journal · 2023Article
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post-translational modifications in the protein's native environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real-time monitoring of protein expression, and also provides an affinity handle for one-step protein purification using a suitable affinity reagent. Here, we describe a panel of anti-GFP and anti-mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target proteins using X-ray crystallography. From these analyses, we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody-sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry-tag system by using it to purify recombinant human topoisomerase 2α expressed in HEK293F cells. The mCherry-tag and GFP/YFP-tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real-time monitoring of protein expression levels and a high-efficiency purification step is needed.
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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.