ArticleJournal of molecular biology2008
The funnel approach to the precrystallization production of membrane proteins.
Article in Journal of molecular biology, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 31 papers.
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Who cites it
31 citing papers in PubMed.
- Protein folding: Funnel model revised.Computational and structural biotechnology journal · 2024Article
- Computational design and molecular dynamics simulations suggest the mode of substrate binding in ceramide synthases.Nature communications · 2023Article
- Biochemical characterization of a GDP-mannose transporter from Chaetomium thermophilum.PloS one · 2023Article
- Dynamics of Co-translational Membrane Protein Integration and Translocation via the Sec Translocon.Journal of the American Chemical Society · 2020Review
- A statistical model for improved membrane protein expression using sequence-derived features.The Journal of biological chemistry · 2018Article
- Improving membrane protein expression by optimizing integration efficiency.The Journal of biological chemistry · 2017Article
- Purification of Ovine Respiratory Complex I Results in a Highly Active and Stable Preparation.The Journal of biological chemistry · 2016Article
- A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency.Cell reports · 2016Article
- Large-scale identification of membrane proteins with properties favorable for crystallization.Protein science : a publication of the Protein Society · 2015Article
- Membrane proteins, detergents and crystals: what is the state of the art?Acta crystallographica. Section F, Structural biology communications · 2014Review
- Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs.Protein expression and purification · 2014Article
- Identification of functionally important conserved trans-membrane residues of bacterial PIB -type ATPases.Molecular microbiology · 2014Article
- A microfluidic approach for protein structure determination at room temperature via on-chip anomalous diffraction.Lab on a chip · 2013Article
- Open and shut: crystal structures of the dodecylmaltoside solubilized mechanosensitive channel of small conductance from Escherichia coli and Helicobacter pylori at 4.4 Å and 4.1 Å resolutions.Protein science : a publication of the Protein Society · 2013Article
- Identification of the dimer interface of a bacterial Ca(2+)/H(+) antiporter.Biochemistry · 2012Article
- Pharmaceutical and biomedical applications of affinity chromatography: recent trends and developments.Journal of pharmaceutical and biomedical analysis · 2012Review
- Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells.Nature protocols · 2012Article
- Efficient expression screening of human membrane proteins in transiently transfected Human Embryonic Kidney 293S cells.Methods (San Diego, Calif.) · 2011Article
- GFP-based evaluation system of recombinant expression through the secretory pathway in insect cells and its application to the extracellular domains of class C GPCRs.Protein science : a publication of the Protein Society · 2011Article
- High throughput platforms for structural genomics of integral membrane proteins.Current opinion in structural biology · 2011Review
Corrections and comments
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Authors and funding
3 authors.
Funding
Abstract
Challenges in the production of integral membrane proteins for structural studies include low expression levels, incorrect membrane insertion, aggregation and instability. In this report, we describe a "funnel approach" to overcoming these difficulties and demonstrate its efficacy in a case study of 36 prokaryotic P-type transporters. A diverse ensemble of modified constructs is generated and tested for expression in Escherichia coli, membrane localization, detergent extraction, and homogeneity. High-throughput methodologies are implemented throughout the process to facilitate identification of promising targets. We find that the choice of promoter, the choice of source organism providing the cloned gene, and, most importantly, the position of the affinity tag have a great effect on successful production. The latter had pronounced effects at all tested levels, from expression levels observed in whole cells to the extent of membrane insertion, and even on protein function. Following the initial streamlined screening, we were able to fine-tune and produce 9 of the 36 targets as materials suitable for crystallization or other structural studies.
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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.