ArticleProtein science : a publication of the Protein Society2013
Fluorescent probe for high-throughput screening of membrane protein expression.
Article in Protein science : a publication of the Protein Society, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed, 40 citations in OpenAlex.
- Overcoming the Solubility Problem in E. coli: Available Approaches for Recombinant Protein Production.Methods in molecular biology (Clifton, N.J.) · 2022Review
- The high-throughput production of membrane proteins.Emerging topics in life sciences · 2021Article
- Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins.Communications biology · 2021Article
- Spectroscopic Analysis of Fe Ion-Induced Fluorescence Quenching of the Green Fluorescent Protein ZsGreen.Journal of fluorescence · 2021Article
- The rapid "teabag" method for high-end purification of membrane proteins.Scientific reports · 2020Article
- Structural basis for oligomerization of the prokaryotic peptide transporter PepTActa crystallographica. Section F, Structural biology communications · 2019Article
- Structural Basis of HStructure (London, England : 1993) · 2019Article
- Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding.Proceedings of the National Academy of Sciences of the United States of America · 2017Article
- MraY-antibiotic complex reveals details of tunicamycin mode of action.Nature chemical biology · 2017Article
- High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives.Open biology · 2016Review
- Article
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Authors and funding
6 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Screening of protein variants requires specific detection methods to assay protein levels and stability in crude mixtures. Many strategies apply fluorescence-detection size-exclusion chromatography (FSEC) using green fluorescent protein (GFP) fusion proteins to qualitatively monitor expression, stability, and monodispersity. However, GFP fusion proteins have several important disadvantages; including false-positives, protein aggregation after proteolytic removal of GFP, and reductions in protein yields without the GFP fusion. Here we describe a FSEC screening strategy based on a fluorescent multivalent NTA probe that interacts with polyhistidine-tags on target proteins. This method overcomes the limitations of GFP fusion proteins, and can be used to rank protein production based on qualitative and quantitative parameters. Domain boundaries of the human G-protein coupled adenosine A2a receptor were readily identified from crude detergent-extracts of a library of construct variants transiently produced in suspension-adapted HEK293-6E cells. Well expressing clones of MraY, an important bacterial infection target, could be identified from a library of 24 orthologs. This probe provides a highly sensitive tool to detect target proteins to expression levels down to 0.02 mg/L in crude lysate, and requires minimal amounts of cell culture.
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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.