Abstract
Abstract To clarify the efficient refolding conditions of enzymes using chaperonin GroE from Escherichia coli , the effects of various factors on the chaperonin-mediated refolding of enzymes from 4M guanidine hydrochloride (GdnHCl) were investigated. Three enzymes, Bacillus subtilis α-amylase, bovine deoxyribonuclease I (DNase I) and yeast enolase were used as the model systems. In all enzymes, the maximum recovery of activities (90–150% with respect to the enzyme activities before denaturation) was attained in the presence of 2mM ATP and 2–5-fold molar excess of GroE 21-mer or GroEL 14-mer over enzyme molecules. Since the recovery of enzyme activity by GroEL is close to that by GroE, GroEL as well as GroE are applicable for the refolding systems of these enzymes. GroE significantly enhanced the recovery of enzyme activities at around 25–40 °C, pH 6–9 and up to rather high final GdnHCl and enzyme concentrations. Therefore, the chaperonin efficiently mediates the enzyme refolding under wide operation conditions. To test the reusability of chaperonins in enzyme refolding, GroEL was separated from refolded enzymes by ultrafiltration and recycled. GroEL repeatedly mediated the refolding of enzymes by choosing appropriate membranes. Therefore, protein refolding processes based on chaperonins are promising.
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