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Protein Expr Purif ; 47(2): 384-92, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16603379

RESUMO

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.


Assuntos
Proteínas de Choque Térmico/isolamento & purificação , Complexos Multiproteicos/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas Arqueais/química , Proteínas Arqueais/ultraestrutura , Citrato (si)-Sintase/química , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/ultraestrutura , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/ultraestrutura , Muramidase/química , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/ultraestrutura
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