sHsps are ubiquitous ATP-independent molecular chaperones, which efficiently prevent the unspecific aggregation of non-native proteins. Here, we described the purification of the small heat shock protein Hsp26 from a Saccharomyces cerevisiae strain harboring a multicopy plasmid carrying HSP26 gene under the control of its native promoter. A 26 kDa protein was purified to apparent homogeneity with a recovery of 74% by a very reproducible three steps procedure consisting of ethanol precipitation, sucrose gradient ultracentrifugation, and heat inactivation of residual contaminants. The purified polypeptide was unequivocally identified as Hsp26 using a specific Hsp26 polyclonal antibody as a probe. The analysis of the purified protein by electron microscopy revealed near spherical particles with a diameter of 12.0 nm (n=57, standard deviation +/-1.6 nm), displaying a dispersion in size ranging from 9.2 to 16.1 nm, identical to Methanococcus jannaschii Hsp16.5 and in the range of the size estimated for yeast Hsp26, in a previous report. Purified yeast Hsp26 was able to suppress 72% of the heat-induced aggregation of citrate synthase at a ratio of 1:1 (Hsp26 24-mer complex to citrate synthase dimer), and 86% of the heat-induced aggregation of lysozyme at a molar ratio of 1:16 (Hsp26 24-mer complex to lysozyme monomer). In conclusion, the Hsp26 protein purified as described here has structure and activity similar to the previously described preparations. As advantages, this new protocol is very reproducible and requires simple apparatuses which are found in all standard biochemistry laboratories.
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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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