ArticleJournal of structural and functional genomics2004
Expression screening, protein purification and NMR analysis of human protein domains for structural genomics.
Article in Journal of structural and functional genomics, 2004. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed.
- Article
- Multi-head attention-based U-Nets for predicting protein domain boundaries using 1D sequence features and 2D distance maps.BMC bioinformatics · 2022Article
- High-Performance Deep Learning Toolbox for Genome-Scale Prediction of Protein Structure and Function.Workshop on Machine Learning in HPC Environments. Workshop on Machine Learning in HPC Environments · 2021Article
- CombinedStructure (London, England : 1993) · 2018Article
- Efficient switching of mCherry fluorescence using chemical caging.Proceedings of the National Academy of Sciences of the United States of America · 2017Article
- PAT: predictor for structured units and its application for the optimization of target molecules for the generation of synthetic antibodies.BMC bioinformatics · 2016Article
- Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling.Journal of biomolecular NMR · 2015Article
- Article
- The Fanconi anemia associated protein FAAP24 uses two substrate specific binding surfaces for DNA recognition.Nucleic acids research · 2013Article
- Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success.Acta crystallographica. Section F, Structural biology and crystallization communications · 2011Article
- DoBo: Protein domain boundary prediction by integrating evolutionary signals and machine learning.BMC bioinformatics · 2011Article
- Mathematical model for empirically optimizing large scale production of soluble protein domains.BMC bioinformatics · 2010Article
- Advances in Nuclear Magnetic Resonance for Drug Discovery.Expert opinion on drug discovery · 2009Article
- Parallel screening and optimization of protein constructs for structural studies.Protein science : a publication of the Protein Society · 2009Article
- The full-ORF clone resource of the German cDNA Consortium.BMC genomics · 2007Article
- Analysis of the XPA and ssDNA-binding surfaces on the central domain of human ERCC1 reveals evidence for subfunctionalization.Nucleic acids research · 2007Article
- Enzyme free cloning for high throughput gene cloning and expression.Journal of structural and functional genomics · 2006Article
- Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination.Protein science : a publication of the Protein Society · 2006Article
- Rapid determination of protein solubility and stability conditions for NMR studies using incomplete factorial design.Journal of biomolecular NMR · 2006Article
- Understanding the relationship between the primary structure of proteins and its propensity to be soluble on overexpression in Escherichia coli.Protein science : a publication of the Protein Society · 2005Article
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Authors and funding
3 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Structural genomics, the determination of protein structures on a genome-wide scale, is still in its infancy for eukaryotes due to the number and size of their genes. Low protein expression and solubility of eukaryotic geneproducts are the major bottlenecks in high-throughput (HTP) recombinant protein production with the E. coli expression systems. To circumvent this problem we decided to focus on separate protein domains. We describe here a fast microtiterplate based, expression and solubility screening procedure, using a combination of in vitro and in vivo expression, and purification with nickel-NTA magnetic beads. All steps are optimized for automatic HTP processing using a liquid handling station. Furthermore, large-scale expression and protein purification conditions are optimized, permitting the purification of 24 protein samples per week. We further show that results obtained from the expression screening can be extrapolated to the production of protein samples for NMR. Starting with 81 cloned human protein domains, in vivo expression was detected in 54 cases, and from 28 of those milligrams of protein were purified. An informative HSQC spectrum was recorded for 18 proteins (22%), half of which were indicative of a folded protein. The success rate and quality of the HSQC spectra suggest that the domain approach holds promise for human proteins.
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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.