ArticleBiochemistry2018
An Improved Strategy for Fluorescent Tagging of Membrane Proteins for Overexpression and Purification in Mammalian Cells.
Article in Biochemistry, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 45 papers.
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Who cites it
45 citing papers in PubMed, 67 citations in OpenAlex.
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- Highly coherent organic lasing based on etch-free metasurface.Nature communications · 2025Article
- Mechanisms of ligand recognition and channel opening for P2X2 receptors in lipid nanodiscs.bioRxiv : the preprint server for biology · 2025Article
- Structural basis for Porcupine inhibition.Communications chemistry · 2025Article
- Multigenerational proteolytic inactivation of restriction upon subtle genomic hypomethylation in Pseudomonas aeruginosa.Nature microbiology · 2025Article
- Structural basis of fast N-type inactivation in KNature · 2025Article
- Effect of recombinant protein production and release on microalgal fitness and the impact of environmental conditions for localized therapeutic delivery.Journal of biological engineering · 2025Article
- PEG-mCherry interactions beyond classical macromolecular crowding.Protein science : a publication of the Protein Society · 2025Article
- Molecular basis of Spns1-mediated lysophospholipid transport from the lysosome.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Structures of synaptic vesicle protein 2A and 2B bound to anticonvulsants.Nature structural & molecular biology · 2024Article
- Transport mechanism and structural pharmacology of human urate transporter URAT1.Cell research · 2024Article
- Discovery of Therapeutic Antibodies Targeting Complex Multi-Spanning Membrane Proteins.BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy · 2024Review
- In vitro reconstitution of transition metal transporters.The Journal of biological chemistry · 2024Review
- Cryo-EM reveals that iRhom2 restrains ADAM17 protease activity to control the release of growth factor and inflammatory signals.Molecular cell · 2024Article
- Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding.Protein science : a publication of the Protein Society · 2024Article
- Eukaryotic Kv channel Shaker inactivates through selectivity filter dilation rather than collapse.Science advances · 2023Article
- Production and Purification of Cysteine-Rich Leptospiral Virulence-Modifying Proteins with or Without mCherry Fusion.The protein journal · 2023Article
- Combined Computational-Biochemical Approach Offers an Accelerated Path to Membrane Protein Solubilization.Journal of chemical information and modeling · 2023Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
An essential prerequisite for in vitro biochemical or structural studies is a construct that is amenable to high level expression and purification and is biochemically "well-behaved". In the field of membrane protein research, the use of green fluorescent protein (GFP) to monitor and optimize the heterologous expression in different hosts has radically changed the ease of streamlining and multiplexing the testing of a large number of candidate constructs. This is achieved by genetically fusing the fluorescent proteins to the N- or C-terminus of the proteins of interest to act as reporters which can then be followed by methods such as microscopy, spectroscopy, or in-gel fluorescence. Nonetheless, a systematic study on the effect of GFP and its spectral variants on the expression and yields of recombinant membrane proteins is lacking. In this study, we genetically appended four common fluorescent protein tags, namely, mEGFP, mVenus, mCerulean, and mCherry, to the N- or C-terminus of different membrane proteins and assessed their expression in mammalian cells by fluorescence-detection size exclusion chromatography (FSEC) and protein purification. We find that, of the four fluorescent proteins, tagging with mVenus systematically results in higher expression levels that translates to higher yields in preparative purifications, thus making a case for switching to this yellow spectral variant as a better fusion tag.
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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.