Abstract
The human inducible heat shock protein 70 (hHsp70), which is involved in several major pathologies, including neurodegenerative disorders and cancer, is a key molecular chaperone and contributes to the proper protein folding and maintenance of a large number of protein structures. Despite its role in disease, the current structural knowledge of hHsp70 is almost exclusively based on its Escherichia coli homolog, DnaK, even though these two proteins only share ~50 % amino acid identity. For the first time, we describe a complete heterologous production and purification strategy that allowed us to obtain a large amount of soluble, full-length, and non-tagged hHsp70. The protein displayed both an ATPase and a refolding activity when combined to the human Hsp40. Multi-angle light scattering and bio-layer interferometry analyses demonstrated the ability of hHsp70 to homodimerize. The role of the C-terminal part of hHsp70 was identified and confirmed by a study of a truncated version of hHsp70 that could neither dimerize nor present refolding activity.Electronic supplementary materialThe online version of this article (doi:10.1007/s12192-014-0526-3) contains supplementary material, which is available to authorized users.
Highlights
Molecular chaperones assist in the non-covalent folding or unfolding of a polypeptide chain, refolding of misfolded intermediates, the assembly or disassembly of a complex, and its proper localization in the cell (Ellis 1987; Fink 1999; Taipale et al 2010)
The Human Hsp70 (hHsp70) is divided into two functional domains connected by a short linker: an amino-terminal nucleotide binding domain (NBD) and a carboxyl-terminal substrate binding domain (SBD)
A synthetic, codon-optimized coding sequence for hHsp70 expression was introduced in a pET21a plasmid
Summary
Molecular chaperones assist in the non-covalent folding or unfolding of a polypeptide chain, refolding of misfolded intermediates, the assembly or disassembly of a complex, and its proper localization in the cell (Ellis 1987; Fink 1999; Taipale et al 2010). Present at low or undetectable levels in most unstressed normal cells and tissues, Hsp ( referred to as Hsp, Hsp, or HspA1A) is among the most stressinducible molecular chaperones (Dworniczak and Mirault 1987; Freeman et al 1995) This ~70 kDa chaperone interacts with Hsp to accommodate its substrates, binds to short linear stretches of hydrophobic residues and refolds its substrate properly through cycles of ATP binding, hydrolysis, and release (Ellis et al 2007; Schlecht et al 2011; Taipale et al 2010). A few independent studies have revealed a mixture of monomeric and dimeric forms after purification, but the in vivo relevance of these forms is a debatable point (Palleros et al 1993; Richarme and Kohiyama 1993)
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