The gene coding for APG8a (At4g21980), a protein from Arabidopsis thaliana, is involved in the autophagy process. The protein is an interesting candidate for structure determination by NMR spectroscopy. Toward this end, APG8a fused to an N-terminal His-tag has been expressed in Escherichia coli under a T7 expression system, refolded in vitro, and kept soluble by slight destabilization. The expressed protein appeared in both the soluble and the insoluble fractions. The whole-cell lysate was denatured by the addition of guanidinium chloride. The protein was immobilized on nickel-agarose resin and refolded by stepwise decrement of the denaturant. The elution buffer was 20 mM sodium phosphate, pH 7.0, with 1% glycerol, 0.5 M urea, 300 mM NaCl, and 1 M imidazole. After the removal of imidazole by ultrafiltration, the His-tag was cleaved with biotinylated thrombin. The protein product was kept in 20 mM sodium phosphate, pH 7.0, with 1% glycerol, 0.5 M urea, and 300 mM NaCl. The protein was found to aggregate extensively over time if any one of the three ingredients (sodium chloride, urea, or glycerol) was omitted. The yield of the protein was around 20 mg/L Luria-Bertani culture medium. The (1)H-(15)N NMR correlation spectrum of (15)N-labeled APG8a showed the characteristic signature of a folded protein; thus, the solutes appear to have no deleterious effect on the sample. These solution conditions kept the protein soluble and unaggregated for at least 2 days (enough time for NMR data collection). This approach of balanced stabilization-destabilization may offer a general approach for structural investigations of proteins that tend to aggregate.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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