Optimization of expression and purification of human mortalin (Hsp70): Folding/unfolding analysis

Khan, M.S. and Ahmed, A. and Tabrez, S. and Islam, B.U. and Rabbani, N. and Malik, A. and Ismael, M.A. and Alsenaidy, M.A. and Alsenaidy, A.M. (2017) Optimization of expression and purification of human mortalin (Hsp70): Folding/unfolding analysis. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy, 187. pp. 98-103. ISSN 13861425

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Abstract

Human mortalin is a Hsp70 mitochondrial protein that plays an essential role in the biogenesis of mitochondria. The deregulation of mortalin expression and its functions could lead to several age-associated disorders and some types of cancers. In the present study, we optimized the expression and purification of recombinant human mortalin by the use of two-step chromatography. Low temperature (18 °C) and 0.5 mM (IPTG) was required for optimum mortalin expression. Chaperone activity of mortalin was assessed by the citrate synthase and insulin protection assay, which suggested their protective role in mitochondria. Folding and unfolding assessments of mortalin were carried out in the presence of guanidine hydrochloride (GdnHCl) by intrinsic fluorescence measurement, ANS (8-analino 1-nephthlene sulfonic acid) binding and CD (circular dichroism) analysis. Under denaturing conditions, mortalin showed decrease in tryptophan fluorescence intensity along with a red shift of 11 nm. Moreover, ANS binding studies illustrated decrease in hydrophobicity. CD measurement of mortalin showed a predominant helical structure. However, the secondary structure was lost at low concentration of GdnHCl (1 M). We present a simple and robust method to produce soluble mortalin and warranted that chaperones are also susceptible to unfolding and futile to maintain protein homeostasis.

Item Type: Article
Uncontrolled Keywords: CD; Chaperone; Intrinsic fluorescence; Mortalin
Subjects: R Medicine > R Medicine (General)
Divisions: Faculties > Faculty of Life Sciences > Department of Biochemistry
Depositing User: AMU Library
Date Deposited: 29 Jan 2018 07:01
Last Modified: 01 Feb 2018 05:10
URI: http://ir.amu.ac.in/id/eprint/11007

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