Heikkila John J, Kaldis Angelo, Abdulle Rashid
Department of Biology, University of Waterloo, Ontario, Canada.
Methods Mol Biol. 2006;322:213-22. doi: 10.1007/978-1-59745-000-3_15.
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.
热休克蛋白（Hsps）是分子伴侣，在正常条件下有助于蛋白质的折叠和转运，并在应激情况下保护细胞蛋白质。一类热休克蛋白，即小分子热休克蛋白，可以使变性的靶蛋白保持在具有折叠能力的状态，以便在其他分子伴侣存在的情况下它们能够重新折叠并恢复生物活性。以往的分析采用细胞裂解物作为参与折叠的分子伴侣的来源。在本章中，我们描述了非洲爪蟾重组小分子热休克蛋白Hsp30C的生产和纯化，以及利用显微注射的非洲爪蟾卵母细胞进行的体内荧光素酶（LUC）重折叠分析。该分析测试当荧光素酶在小分子热休克蛋白或其他分子伴侣存在下热变性时，是否能保持在具有折叠能力的状态。例如，单独将热变性的荧光素酶显微注射到卵母细胞中，酶活性的再激活程度极小。然而，在Hsp30C存在下热变性的荧光素酶在显微注射后酶活性可100%恢复。与体外重折叠分析相比，体内卵母细胞重折叠系统更敏感，所需的分子伴侣更少。此外，该方案不限于测试非洲爪蟾的分子伴侣，因为来自其他生物体的小分子热休克蛋白已成功使用。
