Recombinant expression and in vitro refolding of the yeast small heat shock protein Hsp42.
Small Hsps represent a variation on the theme of protection of proteins from irreversible aggregation by reversible interaction with chaperone proteins. While different sHsps are highly heterogeneous in sequence and size, the common trait is the presence of a conserved alpha-crystallin domain. In addition sHsps assemble into large oligomeric complexes where dimers represent the basic building blocks. Hsp42, a member of the sHsp family in the cytosol of S. cerevisiae, forms ordered oligomers with a barrel-like structure. Here, we present the recombinant expression and purification of Hsp42. We demonstrate, that Hsp42 is expressed in inclusion bodies and can be resolubilized and folded to correct, active oligomers. This indicates that in contrast to thermal unfolding, the chemical disassembly and unfolding of Hsp42 is fully reversible. In comparison to the purification of mature Hsp42 from yeast, its recombinant expression leads to a substantial increase in the yield of the protein and to a reduction of contamination caused by aggregation prone proteins complexed by Hsp42. In addition, the recombinant Hsp42 is fully active as a chaperone in an energy independent manner.
Department Chemie, Lehrstuhl Biotechnologie, Technische Universität München, D-85747 Garching, Germany. martin.haslbeck@ch.tum.de
Martin
M
Haslbeck
D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004734:Energy Metabolism; D004926:Escherichia coli; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002479:Inclusion Bodies / Q000201:enzymology; D018832:Molecular Chaperones / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism
C098005:HSP42 protein, S cerevisiae; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013884:Thiosulfate Sulfurtransferase