Analysis of molecular chaperones using a Xenopus oocyte protein refolding assay.
Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of protein under normal conditions and protect cellular proteins during stressful situations. A family of Hsps, the small Hsps, can maintain denatured target proteins in a folding-competent state such that they can be refolded and regain biological activity in the presence of other molecular chaperones. Previous assays have employed cellular lysates as a source of molecular chaperones involved in folding. In this chapter, we describe the production and purification of a Xenopus laevis recombinant small Hsp, Hsp30C, and an in vivo luciferase (LUC) refolding assay employing microinjected Xenopus oocytes. This assay tests whether LUC can be maintained in a folding-competent state when heat denatured in the presence of a small Hsp or other molecular chaperone. For example, micro-injection of heat-denatured LUC alone into oocytes resulted in minimal reactivation of enzyme activity. However, LUC heat denatured in the presence of Hsp30C resulted in 100% recovery of enzyme activity after microinjection. The in vivo oocyte refolding system is more sensitive and requires less molecular chaperone than in vitro refolding assays. Also, this protocol is not limited to testing Xenopus molecular chaperones because small Hsps from other organisms have been used successfully.
Heikkila
John J
JJ
Department of Biology, University of Waterloo, Ontario, Canada.
Kaldis
Angelo
A
Abdulle
Rashid
R
D000818:Animals; D050890:HSP30 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008845:Microinjections / Q000295:instrumentation / Q000379:methods; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000235:genetics; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D029867:Xenopus Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014982:Xenopus laevis
D050890:HSP30 Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D029867:Xenopus Proteins; C494391:hsp30 protein, Xenopus