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 0.5mM (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 11nm. 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 (1M). 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.
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•The results suggested that expression of recombinant human mortalin was present in soluble form when treated at 0.5 mM IPTG concentration, incubated overnight at 18 oC for 16 hours.•Our expression procedure eliminated the formation of inclusion bodies usually associated with the expression mortalin and also interferes with its functional characteristics.•Mortalin was able to protect citrate synthetase from thermal denaturation suggesting their role as chaperone in mitochondria.•Chemically unfolded mortalin showed loss of secondary and tertiary structure.•A method is optimized to express human mortalin in soluble form using two-step chromatography and showed that the chaperone activity of mortalin was susceptible to denaturant and unable to maintain protein homeostasis.