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KMID : 0368420150580040236
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Journal of Plant Biology
2015 Volume.58 No. 4 p.236 ~ p.241
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Development of in vitro HSP90 foldase chaperone assay using a GST-fused Real-substrate, ZTL (ZEITLUPE)
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Cha Joon-Yung
Kim Mi-Ri
Kim Woe-Yeon
Kim Min-Gab
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Abstract
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Protein folding is one of the essential and fundamental processes involved in all living organisms wherein Heat-Shock Protein 90 (HSP90) served as a chaperone which plays important function in protein folding and further promotes refolding of denatured proteins. There are over six hundred identified substrates of HSP90 at present; however, there is a need to develop a specific folding assay method to test its in vitro foldase activity in case of difficulty in substrate activity measurement in vitro. In previous studies, it has been reported that HSP90 is necessary for the stabilization of ZEITLUPE (ZTL) which plays in the circadian clock and photomorphogenesis in Arabidopsis (Kim et al. 2011). Rhythmic oscillations of ZTL protein might be caused by denaturation and stabilization, thus, refolding activity of HSP90 is possibly involved in the oscillations of ZTL protein. However, the question whether HSP90 indeed promotes refolding of ZTL or not has not yet identified and therefore needs to be investigated. Recombinant glutathione-S transferase (GST) fused-ZTL in bacteria was produced and purified as the soluble GST-ZTL through treatments of both sarkosyl and Triton-X100 as ionic and nonionic detergents, respectively. Upon heat (45°C) treatment, the GST activity of GST-ZTL decreased significantly. After the 3 h heat-induced denaturation of GST-ZTL, the refolding of denatured GST-ZTL was enhanced upon addition of HSP90 in a dose-dependent manner. The results from this study showed that recombinant GST-fused protein can be possibly used for in vitro protein refolding assay method. Moreover, results revealed that Arabidopsis HSP90 can efficiently refolds the denatured GST-ZTL in vitro.
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KEYWORD
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CDNB assay, Foldase chaperone, HSP90, Soluble expression, Zeitlupe
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